Multivalent glycopeptides that tightly bind to target proteins

ABSTRACT

The invention relates to a glycopolypeptide that includes one or more modified amino acid residues having a sidechain comprising a monosaccharide or an oligosaccharide, wherein the glycopolypeptide binds specifically to a carbohydrate-binding monoclonal antibody with an affinity of less than 100 nM. Immunogenic conjugates that include the glycopolypeptide, and pharmaceutical compositions that include the glycopolypeptide or the immunogenic conjugate are also disclosed. Various method of using the glycopolypeptides, immunogenic conjugates, and pharmaceutical compositions are disclosed, including inducing an immune response, inhibiting viral or bacterial infection, treating a cancerous condition, and detecting a neutralizing antibody.

This application is a national stage application under 35 U.S.C. § 371of PCT Application No. PCT/US2014/068195, filed Dec. 2, 2014, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/910,710, filed Dec. 2, 2013, which is hereby incorporated byreference in its entirety.

This invention was made with government support under R01 AI090745awarded by National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to a method for in vitro selection ofglycopeptides that mimic native glycosylated epitopes, the glycopeptidesobtained following such selection and immunogenic conjugates containingthe same, compositions containing these products, and their use toinduce immune responses against the glycopeptides.

BACKGROUND OF THE INVENTION

Antibody 2G12, isolated from an HIV positive individual, binds andneutralizes a broad range of HIV strains (Trkola et al., J. Virol.70:1100-1108 (1996) and Binley et al., J. Virol. 78:13232-13252 (2004))and provides sterilizing immunity against SHIV challenge in macaquemodels of infection (Mascola et al., Nat. Med. 6:207-210 (2000); Hessellet al., Nat. Med. 15:951-954 (2009); and Hessel et al., PLoS Pathog.5:e1000433 (2009)). 2G12 recognizes an epitope comprised of 2-4 highmannose (Man₉GlcNAc₂) glycans on the surface of HIV envelope proteingp120 (Scanlan et al., J. Virol. 76:7306-7321 (2002); Calarese et al.,Science 300:2065-2071 (2003); and Calarese et al. Proc. Natl. Acad. Sci.U.S.A. 102:13372-13377 (2005)) and glycopeptides which precisely mimicthis glycan clustering and presentation may be useful as vaccines to“re-elicit” 2G12-like antibodies in vivo (Scanlan et al., Nature446:1038-1045 (2007)). Glycans clustered on carbohydrate scaffolds (Niet al., Bioconjugate Chem. 17:493-500 (2006)), peptide scaffolds (Joyceet al., Proc. Natl. Acad. Sci. U.S.A. 105:15684-15689 (2008)), andprotein scaffolds (Astronomo et al., J. Virol. 82:6359-6368 (2008)) aswell as phage particles (Astronomo et al., Chem. Biol. 17:357-370(2010)) and yeast (Luallen et al., J. Virol. 82:6447-6457 (2008);Luallen et al., J. Virol. 83:4861-4870 (2009); Agrawal-Gamse et al., J.Virol. 85:470-480 (2011); Ciobanu et al., Chem. Commun. 47:9321-9323(2011); and Marradi et al., J. Mol. Biol. 410:798-810 (2011)) have beentested for this purpose, but with little success. In part, this may bedue to the difficulty of designing structures in which the clustering ofglycans faithfully mimics that of the 2G12 epitope on gp120. Indeed,most of these structures were recognized by 2G12 with orders ofmagnitude weaker affinity than was gp120, suggesting that they were notoptimal mimics of the 2G12 epitope.

The directed evolution of glycopeptides has been of interest, giventheir relevance in both HIV and cancer vaccine design. Although manypowerful methods are available for in vitro selection of peptides,comparatively little has yet been published on in vitro selection ofglycopeptides. Recently phage display with chemically-modified phagesenabled selection of peptide 5-mer sequences containing a single centralmannose monosaccharide from ˜10⁶ sequences (Arai et al., Bioorg. Med.Chem. Lett. 23:4940-4943 (2013)). In an alternative approach, a singlemannose was chemically attached to the N-terminal position of a 7-merphage-displayed library of ˜10⁸ sequences, although selections with thislibrary have not yet been reported (Ng et al., ACS Chem. Biol.7:1482-1487 (2012)). Because carbohydrate epitopes of various pathogen(e.g., HIV) and cancer cells may contain multiple glycans (see Scanlanet al., J. Virol. 76:7306-7321 (2002); Calarese et al., Science300:2065-2071 (2003); and Calarese et al. Proc. Natl. Acad. Sci. U.S.A.102:13372-13377 (2005)), it is desirable that a selection method allowaccess to multivalent glycopeptides containing one or more glycans atvariable positions, supported by a peptide framework.

More importantly, it is desirable for selected glycopeptides to exhibithigh affinity binding to known carbohydrate-binding monoclonalantibodies or other targets used during selection. Suchcarbohydrate-binding monoclonal antibodies include antibodies known toneutralize pathogens and antibodies known to afford protection (i.e.,cytotoxicity) against cancer cells.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the inventions relates to a glycopolypeptide thatincludes one or more modified amino acid residues having a sidechaincomprising a monosaccharide or an oligosaccharide, wherein theglycopolypeptide binds specifically to a carbohydrate-binding monoclonalantibody with an affinity of less than 100 nM.

In particular embodiments, the carbohydrate-binding monoclonal antibodyis a neutralizing antibody that protects against a pathogen, e.g., avirus or bacteria that includes a glycosylated epitope to which theneutralizing antibody binds.

In alternative embodiments, the carbohydrate-binding monoclonal antibodyis a cytotoxic antibody that is cytotoxic to cancer cells that express aglycosylated polypeptide epitope that is unique to cancer cells and towhich the cytotoxic antibody binds.

Certain embodiments according to the first aspect of the inventionrelate to a glycopolypeptide that includes from three to five modifiedamino acid residues having a sidechain comprising a branchedoligosaccharide containing 9 mannose moieties, wherein theglycopolypeptide binds specifically to HIV neutralizing monoclonalantibody 2G12 with an affinity that is substantially the same as orlower than the affinity of the 2G12 antibody to HIV-1 gp120.

A second aspect of the invention relates to an immunogenic conjugatethat includes a glycopolypeptide according to the first aspect of theinvention covalently or non-covalently bound to an immunogenic carriermolecule.

A third aspect of the invention relates to a pharmaceutical compositionthat includes a pharmaceutically acceptable carrier and aglycopolypeptide according to the first aspect of the invention or animmunogenic conjugate according to the second aspect of the invention.

A fourth aspect of the invention relates to a method of inducing animmune response in an individual. This method includes administering toan individual a glycopolypeptide according to the first aspect of theinvention, an immunogenic conjugate according to the second aspect ofthe invention, or a pharmaceutical composition according to the thirdaspect of the invention, wherein the administering is effective toinduce an immune response against the glycopolypeptide.

A fifth aspect of the invention relates to a method of inhibiting viralor bacterial infection that includes: administering to an individual anglycopolypeptide according to the first aspect of the invention, animmunogenic conjugate according to the second aspect of the invention,or a pharmaceutical composition according to the third aspect of theinvention, wherein the administering is effective to induce aneutralizing immune response against a virus or bacterial pathogen.

A sixth aspect of the invention relates to a method of treating acancerous condition that includes administering to an individual aglycopolypeptide according to the first aspect of the invention or apharmaceutical composition according to the second aspect of theinvention, wherein said administering is effective to induce ananti-tumor immune response against a cancer cell expressing aglycosylated cancer-specific protein.

A seventh aspect of the invention relates to a method for detecting aneutralizing antibody in serum that includes providing aglycopolypeptide according to the first aspect of the invention;contacting the glycopolypeptide with serum from an individual; anddetecting whether the glycopolypeptide binds specifically to an antibodypresent in the serum, wherein said detecting is carried out using alabel.

As demonstrated by the accompanying Examples, the selection methoddisclosed herein allows for the generation of a pool of multivalentglycopeptides containing several glycans at variable positions,supported by a significant peptide framework. This selection methodincludes Click chemistry glycosylation of mRNA-displayed peptidelibraries of ˜10¹³ sequences. The usefulness of this selection method isdemonstrated for HIV antigen design, whereby multiple glycopolypeptidescontaining 3-5 high-mannose nonasaccharides were generated and theseglycopolypeptides tightly recognize the broadly neutralizing HIVantibody 2G12. These glycopolypeptides bound to 2G12 with an affinitysubstantially the same as the affinity between 2G12 and HIV-1 gp120.Multiple glycopolypeptides exhibited K_(D)'s below 5 nM, with the bestbinding glycopolypeptide having a K_(D) as low as 500 pM. As a result,these glycopolypeptides adequately mimic the native gp120 epitope, andshould therefore be useful as a vaccine to induce a neutralizing immuneresponse against HIV-1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates how glycopeptide selection can be achieved by thecombination of chemical synthesis, “click: chemistry (CuAAAC, or CopperAssisted Azide Alkyne Cycloaddition) (Kolb et al., Angew. Chem. Int. Ed.40:2004-2021 (2001) and Rostovtsev et al., Angew. Chem. Int. Ed.41:2596-2599 (2002), which are hereby incorporated by reference in theirentirety), mRNA display selection (Roberts et al., Proc. Natl. Acad.Sci. U.S.A. 94:12297-12302 (1997), which is hereby incorporated byreference in its entirety) and codon reassignment (van Hest et al., J.Am. Chem. Soc. 122:1282-1288 (2000) and Tan et al., J. Am. Chem. Soc.126:12752-12753 (2004), which are hereby incorporated by reference intheir entirety) using PURE system cell-free translation (Shimizu et al.,Nat Biotech 19:751-755 (2001); Josephson et al., J. Am. Chem. Soc.127:11727-11735 (2005); Shimizu et al., Methods 36:299-304 (2005);Hartman et al., PLoS ONE 2:e972 (2007); and Guillen et al., J. Am. Chem.Soc. 134:10469-10477 (2012), which are hereby incorporated by referencein their entirety).

FIGS. 2A-D illustrate the in vitro selection of glycopeptides. FIG. 2Ais a schematic illustration of the covalent linkage of nascent peptideto its mRNA, mediated by attachment to mRNA-linked puromycin inside theribosome. FIG. 2B illustrates the use of PURE system to incorporatealkynes via the AUG codon and CuAAAC “click” chemistry glycosylationwith the synthetic Man₉-azide. FIG. 2C illustrates the peptide librariesused in this study. The “fixed” library contains 3 constantglycosylation sites, whereas the “variable” library contains only oneconstant glycosylation site, at position 1. The random regions of bothlibraries are followed by a flexible linker and a His₆ tag. Puromycinattached to mRNA is covalently linked to C-terminal arginine residues intranslation (Josephson et al., J. Am. Chem. Soc. 127:11727-11735 (2005),which is hereby incorporated by reference in its entirety). FIG. 2Dillustrates the scheme for selection of 2G12-binding glycopeptides. Thelibrary DNA is comprised of T7 promotor (P_(T7)), ε-enhancer followed byShine-Dalgarno sequence (SD), the open reading frame (ORF) of thepeptide and the constant region including the sequence for annealing andphoto-crosslinking the mRNA to a puromycin-containing oligonucleotide.

FIG. 3A-C illustrates the examination of the integrity of “click”glycosylated mRNA-peptide fusions before or after reverse transcription.FIG. 3A is an alignment of the peptide sequences for clones 8E and 12G(SEQ ID NOS: 61, 46). The sequences were obtained by cloningnon-selected library DNA. FIG. 3B is an SDS-PAGE analysis of the reversetranscribed mRNA-peptide fusions labeled with ³⁵S-cysteine using a 7.5%precast gel (Bio-Rad). In this condition, cDNA-mRNA-peptide fusionsmigrate faster than mRNA-peptide fusions. FIG. 3C is a SDS-PAGE analysisof fusion integrity. mRNA-peptide fusions were reverse transcribed after(A) or before (B) click reaction. The click reaction was done using aslightly different condition described in the Examples, in which 30-40nM fusions shown in FIG. 3B were incubated with 100 mM HEPES-KOH (pH7.6), 0.02% Triton X-100 (v/v), 1 mM CuSO₄, 2 mM THPTA, 5 mMaminoguanidine hemisulfate and 5 mM sodium ascorbate in the presence (+)or absence (−) of 0.5 mM Man₉-azide, which is a lower concentrationcompared to the regular condition described in the Examples, inargon-filled microtube at room temperature for 3 hours. The fusionswithout click reaction (−) were incubated with 100 mM HEPES-KOH (pH7.6), 0.09% Triton X-100 (v/v) at room temperature in argon-filledmicrotubes for 3 hours as well. The fusions with or without nuclease P₁digestion were separated by 10% Criterion XT Bis-Tris precast gel(Bio-Rad) with XT MES Running Buffer (Bio-Rad). The gels were analyzedby autoradiography.

FIGS. 4A-D illustrate the thermostability of 2G12 and cDNA-mRNA duplexof the library fusions. FIG. 4A is an analysis of the interactionbetween 2G12 and gp120 using BLitz with Dip and Read Ni-NTA Biosensors(ForteBio). 25 μg/ml His₆-tagged gp120 (HIV-1 JRFL) (Immune Technology)was loaded on equilibrated Ni-NTA sensors for 3 min and then the sensorswere equilibrated with selection buffer for 30 s. The 120-loaded sensorswere used to associate with the native 2G12 or the 2G12 heated at 70° C.or 95° C., chilled on ice for 5 min and incubated at room temperature inselection buffer before loading. The time of the 2G12 association anddissociation steps in selection buffer were 2 min. FIG. 4B is a silverstained SDS-PAGE gel in which the 1^(st) supernatant, 2^(nd)supernatant, and bead bound fractions were analyzed. 100 nM 2G12 wasincubated with 6 mg/ml protein G magnetic beads in selection buffer for1 hr and the supernatant was removed (1st sup). The beads wereresuspended in selection buffer and heated at 70° C. for 30 min, chilledon ice for 5 min, and incubated at room temperature for 10 min. Then,the supernatant (2nd sup) was removed and the 2G12 bound to the beadswere eluted out by boil in Laemmli sample buffer (bead bound). Thesupernatants and bead bound fraction were analyzed with the controls ofamounts of input to the beads using 4-20% SDS-PAGE without addition ofreducing agent. The gel was silver-stained. FIG. 4C is the sameexperiment as shown in FIG. 4B using 12 mg/ml protein A magnetic beadsexcept that the incubation time at 70° C. was 20 min. FIG. 4D is anSDS-PAGE gel analyzed by autoradiography. The ³⁵S-cysteine labeledlibrary fusions (50 fmol) for selection round 1 were heated at 95° C.for 2 min or 70° C. for 20 min and chilled on ice before being appliedto 7.5% SDS-PAGE.

FIGS. 5A-B illustrate the experimental results of an in vitro selectionof glycopeptides which bind to HIV broadly neutralizing antibody 2G12.FIG. 5A is a bar graph illustrating the selection conditions andpercentage of radioactivity (counts per minute) in eluted fractions.Concentrations of the 2G12 listed for the selection are prior toaddition of protein G or protein A magnetic beads. FIG. 5B is anSDS-PAGE gel illustrating the profiling of the distribution of theputative number of glycans in library peptides before selection (“n” onthe right on the gel). The boxes indicate enrichment of low-valentglycopeptides in 37° C. selection rounds.

FIGS. 6A-B are SDS-PAGE analyses of the glycosylated peptides selectedin round 10. The numbers “n” on the left of the gel image indicate theputative number of glycans the peptides. In FIG. 6A the peptides werelabeled with ³H-histidine and the bands were visualized by fluorography.In FIG. 6B the peptides were labeled with ³S-cysteine and the bands werevisualized by autoradiography.

FIG. 7 illustrates the binding curves of the interaction between 2G12and selected glycopeptides. The error bars represent standard error.

FIGS. 8A-B illustrate the importance of glycans in binding of selectedglycopeptides to 2G12 and competition with gp120. FIG. 8A is a bar graphshowing the competition of glycopeptide binding to 2G12 with gp120 andmannose and glycosylation-dependent binding. FIG. 8B is a line graphshowing the competition of glycopeptide binding to 2G12 with variedconcentrations of gp120.

FIGS. 9A-C illustrate a binding study of individual glycopeptidesobtained from the starting libraries before selection. FIG. 9A is analignment of the peptide sequences for clones 6E and 12G (SEQ ID NOS:45, 46). The sequences were obtained by cloning non-selected libraryDNA. Both peptides were followed by a flexible linker, a His6-tag and aFLAG-tag (GSGSLGHHHHHHRDYKDDDDK, SEQ ID NO: 1) for purification andradiolabeling purposes. FIG. 9B is an SDS-PAGE analysis of the clickreaction of the non-selected peptides. Both peptides were transcribedand translated from PCR-amplified DNA in Pure System that contains 0.02mg/mL T7 RNA polymerase and additional 1 mM each NTP. The peptides werelabeled with 3H-histidine and the bands were visualized by fluorography.FIG. 9C is a graph of the binding curves for clones 6E and 12G. Datawere obtained as described in Materials and Methods except that the 2G12concentration was 0, 32, 64 or 128 nM and the amount of protein Gmagnetic beads was doubled to accommodate the high 2G12 concentrationsused.

FIGS. 10A-B illustrate results of a BioLayer Interferometry (“BLI”)assay. FIG. 10A is a schematic representation of the preparation ofsynthetic 10F2 glycopeptide and attachment of biotin for immobilizationto streptavidin surface. FIG. 10B is a graph showing the results of aBLI measurement of 2G12 interacting with surface-immobilized synthetic10F2 glycopeptide. K_(on) and k_(off) errors are standard errors of thecurve fit, and the K_(D) error is propagated from those values.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for in vitro selection ofglycopeptides, which involves combining mRNA display with theincorporation of unnatural amino acids and “click” chemistry. Using thisin vitro selection in combination with directed evolution ofglycopeptides, it is possible to develop binding partners with any of avariety of target proteins, including epitope mimics that are boundtightly and specifically by carbohydrate-specific monoclonal antibodies.

Accordingly, the method for selecting a glycopolypeptide that binds to atarget protein includes providing a pool of glycopolypeptides fused viapuromycin linker to an encoding mRNA-cDNA duplex; combining the poolwith a target protein to form a mixture; incubating the mixture for aperiod of time sufficient to allow any target protein to bind to one ormore of the glycopolypeptides, thereby forming glycopolypeptide-targetprotein complexes; and isolating from the mixture theglycopolypeptide-target protein complexes, thereby identifying aplurality of selected glycopolypeptides. Multiple rounds of selectionand regenerating mRNA-linked glycopolypeptide pools can be performed inthe manner illustrated in FIG. 1.

FIG. 1 illustrates how glycopeptide selection can be achieved by thecombination of chemical synthesis, “click” chemistry (CuAAAC, or CopperAssisted Azide Alkyne Cycloaddition) (Kolb et al., Angew. Chem. Int. Ed.40:2004-2021 (2001) and Rostovtsev et al., Angew. Chem. Int. Ed.41:2596-2599 (2002), which are hereby incorporated by reference in theirentirety), mRNA display selection (Roberts et al., Proc. Natl. Acad.Sci. U.S.A. 94:12297-12302 (1997), which is hereby incorporated byreference in its entirety), and codon reassignment (van Hest et al., J.Am. Chem. Soc. 122:1282-1288 (2000) and Tan et al., J. Am. Chem. Soc.126:12752-12753 (2004), which are hereby incorporated by reference intheir entirety) using PURE system cell-free translation (Shimizu et al.,Nat Biotech 19:751-755 (2001); Josephson et al., J. Am. Chem. Soc.127:11727-11735 (2005); Shimizu et al., Methods 36:299-304 (2005);Hartman et al., PLoS ONE 2, e972 (2007); and Guillen et al., J. Am.Chem. Soc. 134:10469-10477 (2012), which are hereby incorporated byreference in their entirety).

The provided pool of glycopolypeptides fused via puromycin linker to anencoding mRNA-cDNA duplex is preferably large enough to affordsufficient diversity so as to allow for selection of multiple, diverseglycopolypeptides that exhibit target protein binding capability. By wayof example, the provided pool comprises about 10¹⁰ or greater, about10¹¹ or greater, about 10¹² or greater, or about 10¹³ or greaterglycopolypeptides fused via puromycin linker to an encoding mRNA-cDNAduplex.

Creation of the first pool is carried out by first generating a libraryof DNA duplexes of sufficient length to afford a glycopeptide pool ofthe desired complexity. Each DNA duplex includes a promoter sequence toallow for transcription, optionally an enhancer element sequence, asequence containing a ribosomal binding site that affords in vitrotranslation of mRNA transcripts, an open reading frame region thataffords sequence variety to generate glycopolypeptide diversity, and adownstream sequence that encodes, e.g., a His tag followed by a constantregion that serves as the linker for puromycin. Any suitable promoterand enhancer sequences suitable for in vitro transcription can be used,and any suitable ribosomal binding sequence can be used. Sequencevariation can be introduced using random diversity at each site orsemi-random diversity at each site.

As shown in FIG. 1 and FIGS. 2A-D, the generation of pools ofmRNA-supported glycopolypeptides and selection of individual poolmembers against target proteins is illustrated. The DNA duplexes areused as templates for generating mRNA templates. This can be achievedusing any suitable in vitro transcription protocol. Thereafter, apuromycin linker is attached to the 3′ region of the mRNA strand.Briefly, purified transcripts can be photo-crosslinked withpuromycin-containing oligonucleotide. Photo-crosslinking is achievedusing, e.g., 365 nm UV irradiation as previously described (Kurz et al.,Nucleic Acids Res. 28:e83 (2000) and Seelig, B. Nat. Protocols 6:540-552(2011), which are hereby incorporated by reference in their entirety).

Use of puromycin at the 3′ region of the mRNA transcript allows formRNA-display of the translated polypeptide based on the physical linkageof the polypeptide to the mRNA that encoded it. Puromycin inhibitstranslation by mimicking the substrate of the ribosome—the 3′ end of anaminoacyl-tRNA. As ribosomes complete the translation of individualmRNAs to the corresponding peptides they encounter the 3′ puromycin.Because puromycin is chemically similar to the 3′ end of aminoacyl-tRNA,it is recognized by the peptidyl transfer center of the ribosome, whichcatalyzes the transfer of the nascent polypeptide to the modifiedtyrosine of puromycin. The mRNA is now covalently attached to thecorresponding translated peptide via the puromycin, and the ribosomesare stalled. To promote the covalent attached (or fusion) of thetranslated polypeptide to the encoding mRNA strand, the reaction mixtureis preferably exposed to KCl and Mg(OAc)₂ and then maintained at atemperature below 0° C. for sufficient duration to yield the fusedproduct. At this point, the initial pool or library mRNAs have now beentranslated and linked via puromycin to the peptides that they encode ina stable molecular conjugate referred to as an mRNA—peptide fusion.

To facilitate glycosylation of the translated polypeptide, translationof the mRNA strand is carried out using one or more modified amino acidscomprising a reactive side chain. One exemplary amino acid ishomopropargylglycine, which is efficiently recognized for incorporationinto the polypeptide corresponding to the location of Met codons. Thus,for purposes of translation, homopropargylglycine constitutes a modifiedmethionine. Homopropargylglycine can be prepared using the procedures ofShimizu et al., Nat Biotech 19:751-755 (2001); Josephson et al., J. Am.Chem. Soc. 127:11727-11735 (2005); Guillen et al., J. Am. Chem. Soc.134:10469-10477 (2012); Shimizu et al., Methods Mol Biol. Vol. 607, p11-21 (2010); and Ma et al., Ribosome Display and Related Technologies;Douthwaite, J. A., Jackson, R. H., Eds.; Springer New York: Methods MolBiol. Vol. 805, p 367-390 (2012), which are hereby incorporated byreference in their entirety. Other exemplary amino acids arep-azido-phenylalanine and p-ethynyl-phenylalanine, which are efficientlyrecognized for incorporation into the polypeptide corresponding to thelocation of Phe codons when the PheRS A294G substrate is used (seeHartmann et al., PlosOne DOI 10.1371/journal.pone.0000972 (2007), whichis hereby incorporated by reference in its entirety. Yet anotherexemplary amino acid is L-allyl glycine which is efficiently recognizedfor incorporation into the polypeptide corresponding to the location ofLeu codons when the editing deficient LeuRS D345A substrate is used.With modified amino acylated-tRNAs introduced into the reaction mixturein the absence of one or more natural amino acylated-tRNAs, the modifiedamino acids are introduced into the polypeptide chain (Guillen et al.,J. Am. Chem. Soc. 134:10469-10477 (2012), which is hereby incorporatedby reference in its entirety).

The resulting translated polypeptide can include any number of aminoacids, preferably between about 10 to about 80 amino acids, morepreferably between about 15 and about 70 amino acids. In certainembodiments, the polypeptide can include about 20 amino acids, about 25amino acids, about 30 amino acids, about 35 amino acids, about 40 aminoacids, about 45 amino acids, about 50 amino acids, about 55 amino acids,about 60 amino acids, or about 65 amino acids. The polypeptide caninclude one or more of the modified amino acid residues, preferablybetween about 2 to about 10 of the modified amino acid residues. Incertain embodiments, the polypeptide can include 2 to 5 modified aminoacids, or 6 to 10 modified amino acids.

The modified amino acids can be located at adjacent positions (i.e.,where one modified amino acid is linked via peptide bond to anothermodified amino acid) or at nonadjacent positions (i.e., where no twomodified amino acids are linked via peptide bond to one another). Incertain embodiments, the resulting polypeptide includes a plurality ofmodified amino acids, some of which are adjacent to one another and someof which are not adjacent to another modified amino acid.

After forming the mRNA-polypeptide fusion, the one or moremonosaccharides or oligosaccharides are attached using appropriate clickchemistry reactions, which include thiol-ene reactions (reaction of athiol bond across an alkene or alkyne by either a free radical or ionicmechanism) (see, e.g., Hoyle et. al., Angew. Chem. Int. Ed. 49:1540-1573(2010), which is hereby incorporated by reference in its entirety) aswell as azide-alkyne cycloaddition reactions (reaction of an azido groupwith a terminal or internal alkyne) (see, e.g., Temme et al., Chem. Eur.J. 19:17291-17295 (2013) and Hong et al., Angew. Chem. Int. Ed.48:9879-9883 (2009), which are hereby incorporated by reference in theirentirety).

The monosaccharide or oligosaccharide to be linked to the modified aminoacid(s) of the polypeptide can be any saccharide modified with a clickchemistry reactive group (e.g., thiol, azide, alkyne or alkene).Suitable monosaccharides include, without limitation glucose, galactose,mannose, arabinose, fucose, rhamnose, sialic acid, andN-acetyl-glucosamine.

Suitable oligosaccharides include branched or unbranched oligosaccharidethat include at least 3 saccharide moieties, typically from about 3saccharide moieties up to about 20 saccharide moieties. The saccharidemoieties include those identified as suitable monosaccharides.

Exemplary N-linked glycan structures include high mannose N-glycanspresent in the human lung:

where saccharide subunits include N-acetylglucosamine and mannose asshown (Walther et al., PLOS Pathogens 9(3):e1003223 (2013), which ishereby incorporated by reference in its entirety).

Exemplary N-linked glycan structures recognized by HIV broadlyneutralizing antibodies (PGT151-PGT158) include multi-antennarycomplex-type N-glycans with terminal galactose with and without sialicacid residues:

where saccharide subunits include N-acetylglucosamine, mannose,galactose, sialic acid, and fucose as shown (Walther et al., PLOSPathogens 9(3):e1003223 (2013) and Falkowska et al., Immunity 40(5):657-6688 (2014), which are hereby incorporated by reference in theirentirety).

Additional exemplary N-linked glycan structures include hybrid-typeglycans recognized by HIV antibody PG16:

where saccharide subunits include N-acetylglucosamine, mannose,galactose, and sialic acid (Pancera et al., Nature Struct Mol Biol.20(7): 804-13 (2013), which is hereby incorporated by reference in itsentirety).

Derivatization of the monosaccharides and/or oligosaccharides tointroduce the reactive azido, alkynyl, alkenyl, or thiol group can beachieved using known procedures. See, e.g., Hoyle et al., Angew. Chem.Int. Ed. 49:1540-1573 (2010); Temme et al., Chem. Eur. J. 19:17291-17295(2013); Hong et al., Angew. Chem. Int. Ed. 48:9879-9883 (2009);MacPherson et al., Angew. Chem. Int. Ed. 50:11238-11242 (2011); Kolb etal., Angew. Chem. Int. Ed. 40:2004-2021 (2001); Rostovtsev et al.,Angew. Chem. Int. Ed. 41:2596-2599 (2002); Gierlich et al., Org. Lett.8:3639-3642 (2006); Gierlich et al., Chem. Eur. J. 13:9486-9494 (2007),each of which is hereby incorporated by reference in its entirety).

Additional exemplary modified oligosaccharides (suitable for clickreaction) include the following:

where A is the mono- or oligosaccharide,

As an alternative to the above structures bearing an azide functionalgroup, equivalent structures can be created with alkynyl, alkenyl, orthiol functional groups.

Tumor-associated carbohydrates (“TACAs”) can be linked to lipids such asgangliosides, or to proteins such as mucins. Exemplary glycolipid TACAsincludes GM2, GD2, GD3, fucosyl-GM1, Globo-H, and Lewis^(y) (Le^(y)) andthe glycoprotein TACAs include the truncated Tn-, TF and sialylated Tn(STn)-antigens as well as Globo-H and Le^(y) (Buscas et al., Chem Commun(Camb). (36): 5335-49 (2009), which is hereby incorporated by referencein its entirety):

These structures can be derivatized to include an azido, alkynyl,alkenyl, or thiol group using the procedures identified above.

An exemplary GPI glycan includes the synthetic non-toxic malarial GPIglycan structureNH₂—CH₂—CH₂—PO₄-(Manα1-2)6Manα1-2Manα-6Manα1-4GlcNH₂α1-6myo-inositol-1,2-cyclic-phosphate(Schofield et al., Nature 418(6899):785-9 (2002), which is herebyincorporated by reference in its entirety):

This structure can be derivatized to include an azido, alkynyl, alkenyl,or thiol group using the procedures identified above.

As a result of the click reaction between the modified amino acid andthe modified monosaccharide or oligosaccharide, the glycopolypeptidecontains a linker molecule between the polypeptide chain and themonosaccharide or oligosaccharide. Exemplary linker molecules include,without limitation:

(resulting from the azide-alkyne reaction) or

(resulting from the alkene/alkyne-thiol reaction), wherein each of R₁and R₂ is optionally a direct link or independently selected from thegroup consisting of a linear or branched C₁ to C₁₈ hydrocarbon that issaturated or mono- or poly-unsaturated, optionally interrupted by one ormore non-adjacent —O—, —C(═O)—, or —NR₄—; a substituted or unsubstitutedC₃ to C₁₀ cycloalkandiyl, a substituted or unsubstituted aryl diradical;a substituted or unsubstituted heteroaryl diradical; a monosaccharidediradical; or a disaccharide diradical; R₃ is optional and can be —O—,—S—, or —NR₄—; and R₄ is H or a C₁ to C₁₀ alkyl.

Although flexible linkers may be used, the linker between themonosaccharide/oligosaccharide and the modified amino acid(s) of theglycopeptide preferably includes or more cyclic moieties which offersome rigidity to the resulting glycosyl group.

After recovering the glycopolypeptide-mRNA fusion, a reversetranscription reaction procedure is performed using the mRNA strand as atemplate to form a cDNA strand. After synthesis of the cDNA strand, theresulting product includes the glycopolypeptide linked to the mRNA-cDNAduplex via puromycin. Collectively, these structures constitute thefirst pool available for selection against a target molecule.

Exemplary target molecules suitable for selection include those thatbind to glycosylated naturally occurring proteins, such as monoclonalantibodies that bind to glycosylated epitopes (i.e.,carbohydrate-binding monoclonal antibodies). Suitablecarbohydrate-binding monoclonal antibodies include those that areneutralizing against a pathogen, as well as those that are cytotoxicagainst a cancer cell.

Exemplary carbohydrate-binding neutralizing monoclonal antibodiesinclude those that bind specifically to N-glycosylated HIV gp120 orN-glycosylated HSV-2 gD. Specific examples of these neutralizingmonoclonal antibodies include, without limitation, 2G12, PG9, PG16,PGT121, PGT122, PGT123, PGT125, PGT126, PGT127, PGT128, PGT129, PGT130,PGT131, PGT135, PGT136, PGT137, PGT141, PGT142, PGT143, PGT144, PGT145,PGT151, PGT152, PGT153, PGT154, PGT155, PGT156, PGT157, PGT158, CH01,CH02, CH03, CH04, 10-1074, 10-996, 10-1146, 10-847, 10-1341, 10-1121,10-1130, 10-410, 10-303, 10-259, 10-1369, and E317.

Exemplary carbohydrate-binding cytotoxic monoclonal antibodies includethose that binds specifically to O-glycosylated cancer-specific humanpodoplanin; aberrantly O-glycosylated cancer-specific MUC1, aberrantlyO-glycosylated cancer-specific Integrin α3β1, or N-glycosylatedcancer-specific antigen RAAG12. Specific examples of these cytotoxicmonoclonal antibodies include, without limitation, LpMab-2 (Kato et al.,Sci Rep. 4:5924 (2014), which is hereby incorporated by reference in itsentirety), 237 MAb (Brooks et al., PNAS 107(22):10056-10061 (2010),which is hereby incorporated by reference in its entirety), RAV12 (Looet al., Mol. Cancer Ther. 6(3):856-65 (2007), which is herebyincorporated by reference in its entirety), BCMab1 (Clinical CancerResearch 20(15):4001 (2014), which is hereby incorporated by referencein its entirety), DF3 and 115D8 (Tang et al., Clin Vaccine Immunol.17(12): 1903-1908 (2010), which is hereby incorporated by reference inits entirety), huHMFG1, HT186-B7, -D11 and -G2 sc-FVs (Thie et al., PLoSOne 6(1): e15921 (2011), which is hereby incorporated by reference inits entirety), and GOD3-2C4 (Welinder et al. Glycobiol. 21(8):1097-107(2011), which is hereby incorporated by reference in its entirety).

Selection of library members that bind to the target protein—in the caseof the monoclonal antibodies, mimicking the native glycosyl-epitope towhich the antibody binds—is carried out in liquid medium. Briefly, thelibrary is introduced into the selection medium with the target protein.If the target protein is biotinylated, streptavidin-labeled magneticbeads can be used to recover library members that bind to the targetprotein. Alternatively, where the target protein is a monoclonalantibody, Protein A or Protein G-labeled magnetic beads can be used torecover library members that bind to the target monoclonal antibody.Regardless of the type of beads used, the beads can be magneticallyisolated and washed with selection buffer. To elute the selected librarymembers, the beads can be resuspended in selection buffer and thenheated to disrupt the affinity binding between library member andtarget. Recovered supernatant contains the eluted library members.

Following recovery of the selected library members, PCR amplification isused to amplify the cDNA portion of the library member mRNA-cDNAduplexes. PCR using Taq DNA polymerase (Roche) is performed usingforward and reverse primers, and the amplified DNAs can be purified andused to regenerate the next selection round. In certain embodiments,error prone PCR can be used to facilitate evolution of the library.

In regenerating the next select round, the transcription, puromycinlinkage, translation, and reverse transcription steps described aboveare used to generate a next generation pool (i.e., the glycopolypeptideslinked mRNA-cDNA duplex via puromycin).

Differences in the selection protocol can performed in subsequentrounds. For instance, the selection stringency can be increased topromote the selection of high affinity binding of pool members. Incertain embodiments the temperature can be varied from about 18 to 22°C. in early rounds to temperatures greater than 22° C. or even greaterthan 27° C. (e.g., about 32° C. to about 42° C.) in later rounds. Anysuch variation in temperature can be used. In alternative embodimentsthe target protein concentration can be varied from about 25 to about200 nM in early rounds, and reduced to about 10 to about 80 nM, or about5 to about 25 nM in later rounds. Any such variation in target proteinconcentration can be used. In certain embodiments the duration of theselection step can also be reduced from about 10 to about 30 minutes inearly rounds, to about 5 to about 20 minutes in later rounds. Any suchvariation in duration of the selection step can be used. In anotherembodiment, the introduction of competitor molecules for negativeselection can be introduced in later rounds, including the introductionof free monosaccharides or oligosaccharides, the introduction ofunglycosylated peptides (removing polypeptides which bind to targetprotein without being glycosylated), the introduction of unmodifiedmagnetic beads, e.g., streptavidin, Protein A, or Protein G-conjugatedbeads (removing polypeptides or glycopolypeptides that bind directly toa solid support), or combinations thereof. Any number of negativeselection steps can be employed. In yet another embodiment, the numberand conditions of the wash steps can be made more stringent during laterselection rounds.

In between rounds or after the final round, the individual, selectedpool members can be sequenced and, thus, the polypeptide sequenceidentified. Having identified the polypeptide sequence, individualglycopolypeptides can be synthesized such that the molecule excludes thepuromycin linker that links the polypeptide sequence to the mRNAtranscript encoding the same. Polypeptide synthesis can be carried outusing, e.g., standard peptide synthesis operations. These include bothFMOC (9-Fluorenylmethyloxy-carbonyl) and tBoc (tert-Butyl oxy carbonyl)synthesis protocols that can be carried out on automated solid phasepeptide synthesis instruments including the Applied Biosystems 431A,433A synthesizers and Peptide Technologies Symphony or large scaleSonata or CEM Liberty automated solid phase peptide synthesizers. Themodified amino acids can be substituted during solid phase synthesis toallow for glycosylation in the same manner as the selectedglycopolypeptides.

The amino acids used during synthesis can be L amino acids, D aminoacids, or a mixture of L and D amino acids. As noted above, the lengthof the glycopolypeptide can be any length, but preferably between about10 to about 80 amino acids.

The glycopolypeptides of the present invention include one or more ofthe modified amino acid residues having a sidechain comprising amonosaccharide or an oligosaccharide, and the glycopolypeptide bindsspecifically to a carbohydrate-binding monoclonal antibody with anaffinity of less than 100 nM.

In certain embodiments, the glycopolypeptide binds specifically to thecarbohydrate-binding monoclonal antibody with an affinity (K_(d)) ofless than 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM,9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.

In preferred embodiments, the glycopolypeptide binds specifically to thecarbohydrate-binding monoclonal antibody with an affinity that issubstantially the same as or lower than the affinity of thecarbohydrate-binding monoclonal antibody to its naturally occurringbinding partner. As used herein, an affinity that is “substantially thesame” means that as Kd of glycopeptide for its target is less than 5×,less than 4×, less than 3×, less than 2×, or less than 1.5×Kd of thenative binding partner to the monoclonal antibody. In certainembodiments, the glycopolypeptide binds specifically to thecarbohydrate-binding monoclonal antibody with an affinity that is lowerthan the affinity of the carbohydrate-binding monoclonal antibody to itsnaturally occurring binding partner.

Exemplary neutralizing monoclonal antibodies and cytotoxic monoclonalantibodies are identified above. Using the selection protocol and thedemonstrated results presented in the accompanying Examples, the presentapplication demonstrates that glycopolypeptides that bind specificallyto carbohydrate-binding monoclonal antibodies can be prepared and it isexpected that these will display higher affinity for the monoclonalantibody than the monoclonal antibody has for its binding partner.

In certain embodiments, the carbohydrate-binding monoclonal antibody isHIV-1 neutralizing monoclonal antibody 2G12 and the glycopolypeptideincludes the sequence XXSIPXYTY (SEQ ID NO: 2) where X at positions 2and 6 is optional and can be any amino acid and X at position 1 is themodified amino acid residue to which the oligosaccharide is linked. Theoligosaccharide consists of a branched Man₉ moiety, which is linked viathe click chemistry linker to the modified amino acids (in oneembodiment that modified amino acid is a modified methionine such ashomopropargyl-glycine).

Exemplary glycopolypeptides containing the consensus sequence of SEQ IDNO: 2 above include, without limitation,

SEQ ID Sequence NO: XDTLHLKQIGGXPNCITQQDVRXTSIPYTYTWP  3XLLKXVDQSRLXPVPGIGVTLHXRSIPYSYLPI  4 XRSTLNSLEYRXQYATEDPRIRXASIPYTYWWP 5 ATKTNCKREKTXDNHVTIXRSIPWYTYRWLPN  6 XATKTNFKREKTXDNHVTIXRSIPWYTYRWLPN 7 XATRTNCKREKTXDNHVTIXRSIPWYTYRWLPN  8XATKTSCKREKTXDNHVTIXRSIPWYTYRWLPN  9 XVLPTIISTNVNPFRXLSIPTYTYLXPITWGEI10 XTSIPYTYLNRSLWTNYRVNSWSXSKNVNVXPL 11XERPSLXCGLSXLTSGGTQSSVXRSIPFYTYWW 12 XATKTNSKREKTXDNHVTIXRSIPWYTYRWLPN53 XATKTNSKREKTXDNHVTIXRSIPWYTYRWLPN 54 XRSIPWYTYRWLPN 55XDTLHLKQIGGXPNSITQQDVRXTSIPYTYTWP 62

In certain embodiments, the carbohydrate-binding monoclonal antibody isHIV-1 neutralizing monoclonal antibody 2G12 but the glycopolypeptidedoes not contain the consensus sequence of XXSIPXYTY (SEQ ID NO: 2) asdefined above. Exemplary glycopolypeptides that do not contain theconsensus include, without limitation:

SEQ ID Sequence NO: XHPYNTSRTSAXXAALKXQVTDXYALALFHRIL 13XSPHLPVLLCKXVLNDGRRIVQXSCELPXVRRS 14 XLXFIRIYPTRXQYVYHAPLLTXVRXSPTGPLI15 XCYVTVIPAXNXPEARLGIVCHXPGIRRGKALY 16XSPHLPVLLSKXVLNDGRRIVQXSSELPXVRRS 52 XXAALKXQVTDXYALALFHRIL 56wherein X is the modified amino residue to which the oligosaccharide islinked, in one embodiment that modified amino acid is a modifiedmethionine such as homopropargylglycine.

More preferred glycopolypeptides are those that bind specifically to the2G12 antibody with a K_(d) value that is lower than 5 nM. Exemplarymembers of this embodiment include, without limitation, the followingsequences:

In certain embodiments, the glycopolypeptide contains from three to fivemodified amino acid residues having a sidechain including a branchedoligosaccharide containing 9 mannose moieties, wherein theglycopolypeptide binds specifically to HIV-1 neutralizing monoclonalantibody 2G12 with an affinity that is substantially the same as orlower than the affinity of the 2G12 antibody to gp120. Antibody 2G12binds to gp120 with an affinity (K_(D)) of 5.8 nM (Hoorelbeke et al., J.FEBS Lett. 587:860-866 (2013), which is hereby incorporated by referencein its entirety).

More preferred glycopolypeptides are those that bind specifically to the2G12 antibody with a K_(d) value that is lower that 5 nM. Exemplarymembers of this embodiment include, without limitation, the followingsequences:

SEQ ID Sequence NO: XLXFIRIYPTRXQYVYHAPLLTXVRXSPTGPLI 15XHPYNTSRTSAXXAALKXQVTDXYALALFHRIL 13 XCYVTVIPAXNXPEARLGIVCHXPGIRRGKALY16 XSPHLPVLLCKXVLNDGRRIVQXSCELPXVRRS 14XLLKXVDQSRLXPVPGIGVTLHXRSIPYSYLPI  4 XDTLHLKQIGGXPNCITQQDVRXTSIPYTYTWP 3 XRSTLNSLEYRXQYATEDPRIRXASIPYTYWWP  5XATKTNCKREKTXDNHVTIXRSIPWYTYRWLPN  6 XTSIPYTYLNRSLWTNYRVNSWSXSKNVNVXPL11 XVLPTIISTNVNPFRXLSIPTYTYLXPITWGEI 10XSPHLPVLLSKXVLNDGRRIVQXSSELPXVRRS 52 XATKTNSKREKTXDNHVTIXRSIPWYTYRWLPN53 XATKTNAKREKTXDNHVTIXRSIPWYTYRWLPN 54 XXAALKXQVTDXYALALFHRIL 56XDTLHLKQIGGXPNSITQQDVRXTSIPYTYTWP 62wherein X is the modified amino residue to which the oligosaccharide islinked, and in one embodiment that modified amino acid is a modifiedmethionine such as homopropargylglycine. Of these, SEQ ID NOs: 13 and 15exhibit the highest affinity with a K_(D) of about 500 to 600 pM.

A further aspect of the invention relates to an immunogenic conjugatethat includes a glycopolypeptide of the invention covalently ornon-covalently bound to an immunogenic carrier molecule. Exemplaryimmunogenic carrier molecule include, without limitation, bovine serumalbumin, chicken egg ovalbumin, keyhole limpet hemocyanin, tetanustoxoid, diphtheria toxoid, thyroglobulin, a pneumococcal capsularpolysaccharide, CRM 197, and a meningococcal outer membrane protein.

Any of a variety of conjugation methodologies can be utilized. See,e.g., Jennings et al., J. Immunol. 127:1011-8 (1981); Beuvery et al.,Infect. Immun 40:39-45 (1993), each of which is hereby incorporated byreference in its entirety. In one approach terminal aldehyde groups canbe generated through periodate oxidation, and the aldehydes are thenreacted through reductive amination with free amino groups on theprotein, mostly lysines, in the presence of sodium cyanoborohydride. Inanother approach, a carbodiimide reaction is performed to covalentlylink carboxylic groups to the lysine ε-amino groups on the carrierprotein. The activation sites in this method are more random, comparedto periodate activation.

A further aspect of the invention relates to a pharmaceuticalcomposition that includes a pharmaceutically acceptable carrier and aglycopolypeptide or immunogenic conjugate of the invention.

Pharmaceutical compositions suitable for injectable or parental use(e.g., intravenous, intra-arterial, intramuscular, etc.) or intranasaluse may include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the form should be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. Suitable adjuvants, carriers and/or excipients,include, but are not limited to sterile liquids, such as water, salinesolutions, and oils, with or without the addition of a surfactant andother pharmaceutically and physiologically acceptable carriers.Illustrative oils are those of petroleum, animal, vegetable, orsynthetic origin, for example, peanut oil, soybean oil, or mineral oil.In general, water, saline, aqueous dextrose and related sugar solutions,and glycols, such as propylene glycol or polyethylene glycol, arepreferred liquid carriers, particularly for injectable solutions.

The pharmaceutical compositions of the present invention may also beadministered directly to the airways in the form of an aerosol. For useas aerosols, the compositions of the present invention in the form of asolution or suspension may be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. The pharmaceutical compositions of the present invention alsomay be administered in a non-pressurized form such as in a nebulizer oratomizer. Formulations suitable for intranasal nebulization or bronchialaerosolization delivery are also known and can be used in the presentinvention (see Lu & Hickey, “Pulmonary Vaccine Delivery,” Exp RevVaccines 6(2):213-226 (2007) and Alpar et al., “BiodegradableMucoadhesive Particulates for Nasal and Pulmonary Antigen and DNADelivery,” Adv Drug Deliv Rev 57(3):411-30 (2005), which are herebyincorporated by reference in their entirety.

The pharmaceutical compositions of the present invention can alsoinclude an effective amount of a separate adjuvant. Suitable adjuvantsfor use in the present invention include, without limitation, aluminumhydroxide, aluminum phosphate, aluminum potassium sulfate, berylliumsulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-wateremulsions, muramyl dipeptide, bacterial endotoxin, lipid, Quil A,non-infective Bordetella pertussis, QS-21, monophosphoryl lipid A, analpha-galactosylceramide derivative, or PamCys-type lipids.

The choice of an adjuvant depends on the stability of the immunogenicformulation containing the adjuvant, the route of administration, thedosing schedule, the efficacy of the adjuvant for the species beingvaccinated, and, in humans, a pharmaceutically acceptable adjuvant isone that has been approved or is approvable for human administration bypertinent regulatory bodies. For example, alum, MPL or IncompleteFreund's adjuvant (Chang et al., Advanced Drug Delivery Reviews32:173-186 (1998), which is hereby incorporated by reference in itsentirety) alone or optionally all combinations thereof are suitable forhuman administration.

The pharmaceutical compositions can also include one or more additivesor preservatives, or both.

Effective amounts of the glycopolypeptide may vary depending upon manydifferent factors, including mode of administration, target site,physiological state of the patient, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. The amount ofglycopolypeptide immunogen depends on whether adjuvant is alsoadministered, with higher dosages being required in the absence ofadjuvant. The amount of a glycopolypeptide immunogen for administrationsometimes varies from 1 μg-5 mg per patient and more usually from 5-1000μg per injection for human administration.

The glycopolypeptides, immunogenic conjugates, and pharmaceuticalcompositions can be incorporated into a delivery vehicle to facilitateadministration. Such delivery vehicles include, but are not limited to,biodegradable microspheres (MARK E. KEEGAN & W. MARK SALTZMAN , SurfaceModified Biodegradable Microspheres for DNA Vaccine Delivery, in DNAVACCINES: METHODS AND PROTOCOLS 107-113 (W. Mark Saltzman et al., eds.,2006), which is hereby incorporated by reference in its entirety),microparticles (Singh et al., “Nanoparticles and Microparticles asVaccine Delivery Systems,” Expert Rev Vaccine 6(5):797-808 (2007), whichis hereby incorporated by reference in its entirety), nanoparticles(Wendorf et al., “A Practical Approach to the Use of Nanoparticles forVaccine Delivery,” J Pharmaceutical Sciences 95(12):2738-50 (2006) whichis hereby incorporated by reference in its entirety), liposomes (U.S.Patent Application Publication No. 2007/0082043 to Dov et al. andHayashi et al., “A Novel Vaccine Delivery System UsingImmunopotentiating Fusogenic Liposomes,” Biochem Biophys Res Comm261(3): 824-28 (1999), which are hereby incorporated by reference intheir entirety), collagen minipellets (Lofthouse et al., “TheApplication of Biodegradable Collagen Minipellets as Vaccine DeliveryVehicles in Mice and Sheep,” Vaccine 19(30):4318-27 (2001), which ishereby incorporated by reference in its entirety), and cochleates(Gould-Fogerite et al., “Targeting Immune Response Induction withCochleate and Liposome-Based Vaccines,” Adv Drug Deliv Rev 32(3):273-87(1998), which is hereby incorporated by reference in its entirety).

The glycopolypeptides, immunogenic conjugates, and pharmaceuticalcompositions can be used to induce an immune response in an individual.The individual can be any mammal, particularly a human, althoughveterinary usage is also contemplated. This method is carried out byadministering one of these active agents to an individual in a mannerthat is effective to induce an immune response against theglycopolypeptide. Because the glycopolypeptide mimics the nativeglycosylated epitope of a native target of the monoclonal antibody towhich the glycopolypeptide was selected, certain glycopolypeptides caninduce a carbohydrate-binding, neutralizing antibody response that isprotective against a pathogen (e.g., viral or bacterial pathogen) andcertain other glycopolypeptides can induce a carbohydrate-binding,cytotoxic antibody response against a cancer cell that expresses aglycosylated antigen.

For each of these embodiments, administration of the glycopolypeptides,immunogenic conjugates, and/or pharmaceutical compositions can becarried orally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, by intranasal instillation, byimplantation, by intracavitary or intravesical instillation,intraarterially, intralesionally, transdermally, intra- orperi-tumorally, by application to mucous membranes, or by inhalation.Administration of these agents can be repeated periodically.

Exemplary viruses include, without limitation, Calicivirus, Chikungunyavirus, Cytomegalovirus, Dengue virus, Eastern Equine Encephalitis virus,Ebola virus, Epstein-Barr virus, Hantaan virus, Hepatitis A virus,Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis Evirus, Herpes simplex virus, Human Immunodeficiency virus (HIV-1 orHIV-2), Human Papillomavirus, Influenza virus, Japanese encephalitisvirus, Junin virus, Lassa virus, Marburg virus, Measles virus,Metapneumovirus, Nipah virus, Newcastle disease virus, Norwalk virus,Parainfluenza virus, Poliovirus, Rabies virus, Respiratory Syncytialvirus, Rift Valley Fever virus, Rotavirus, Rubella virus, Sendai virus,Severe Acute Respiratory Syndrome (SARS Co—V), Tick-borne Encephalitisvirus, Varicella zoster virus, Venezuelan Equine Encephalitis virus,Yellow Fever virus, Western Equine Encephalitis virus, and West Nilevirus.

The use of one or more of the glycopeptides according to SEQ ID Nos:3-16 in an immunogenic conjugate or pharmaceutical composition isspecifically contemplated for prophylactic or therapeutic treatmentagainst HIV-1.

Exemplary bacteria include, without limitation, Bacillus anthracis,Bordetella pertussis B, Borrelia burgdorferi, Chlamydia trachomatis,Clostridium difficile, Clostridium tetani, Candida albicans,Corynebacterium diphtheriae, Cryptococcus neoformans, Entamoebahistolytica, Escherichia coli, Francisella tularensis, Haemophilusinfluenzae (nontypeable), Helicobacter pylori, Histoplasma capsulatum,Moraxella catarrhalis, Mycobacterium leprae, Mycobacterium tuberculosis,Neisseria gonorrheae, Neisseria meningitides, Pseudomonas aeruginosa,Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus,Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcuspyogenes, and Yersinia pestis.

For prophylactic treatment against viral or bacterial infection, it isintended that the glycopolypeptides, immunogenic conjugates, andpharmaceutical compositions of the present invention can be administeredprior to exposure of an individual to the virus or bacteria and that theresulting immune response can inhibit or reduce the severity of theviral or bacterial infection such that the virus or bacteria can beeliminated from the individual. The glycopolypeptides, immunogenicconjugates, and pharmaceutical compositions of the present invention canalso be administered to an individual for therapeutic treatment. Inaccordance with one embodiment, it is intended that the composition(s)of the present invention can be administered to an individual who isalready exposed to the virus or bacteria. The resulting enhanced immuneresponse can reduce the duration or severity of the existing viral orbacterial infection, as well as minimize any harmful consequences ofuntreated viral or bacterial infections. The composition(s) can also beadministered in combination other therapeutic anti-viral oranti-bacterial regimen. In asymptomatic patients, treatment can begin atany age (e.g., 10, 20, 30 years of age). Treatment typically entailsmultiple dosages over a period of time. Treatment can be monitored byassaying antibody, or activated T-cell or B-cell responses to thetherapeutic agent over time. If the response falls, a booster dosage isindicated.

The glycopolypeptides, immunogenic conjugates, and pharmaceuticalcompositions that induce a cytotoxic antibody response against a cancercell antigen can be used to treat solid tumors and blood cancers(leukemia or lymphoma) that are characterized by expression ofO-glycosylated cancer-specific human podoplanin; aberrantlyO-glycosylated cancer-specific MUC1, aberrantly O-glycosylatedcancer-specific integrin α3β1, or N-glycosylated cancer-specific antigenRAAG12.

Exemplary cancers that display one of the glycosylated cancer-specificantigen include colorectal cancer, gastric cancer, ovarian cancer,breast cancer, and pancreatic cancer, which display N-glycosylatedRAAG12; squamous cell carcinoma, lung and esophageal carcinoma,testicular seminoma, malignant brain tumor, fibrosarcoma, malignantmesothelioma, bladder cancers, and testicular cancers that displayO-glycosylated ppodoplanin; bladder cancers that display O-glycosylatedintegrin α3β1; breast cancer, ovarian cancer, lung cancer, pancreaticcancer, prostate cancer, and forms of leukemia that displays aberrantlyO-glycosylated MUC1.

For cancer therapy, it is contemplated that the glycopolypeptides,immunogenic conjugates, and pharmaceutical compositions can beadministered in combination with a chemotherapeutic agent, a radiationtherapy, or alternative immunotherapeutic agent. The specific selectionof chemotherapeutic agent, a radiation therapy, or alternativeimmunotherapeutic agent will depend on the type of cancer. These agentscan also be administered in combination with surgical resection toremove cancerous tissue, with treatment being carried out before, after,or both before and after surgery.

For inducing the immune response, the amount of a glycopolypeptide foradministration sometimes varies from 1 μg-5 mg per patient and moreusually from 5-1500 μg per dose for human administration. Occasionally,a higher dose of 1-2 mg per injection is used. Typically about 10, 20,50, or 100 μg is used for each human dose. The mass of glycopolypeptideimmunogen also depends on the mass ratio of immunogenic epitope withinthe glycopolypeptide immunogen to the mass of glycopolypeptide immunogenas a whole. Typically, 10⁻³ to 10⁻⁵ micromoles of immunogenic epitopeare used for each microgram of glycopolypeptide immunogen. The timing ofinjections can vary significantly from once a day, to once a year, toonce a decade. On any given day that a dosage of glycopolypeptideimmunogen is given, the dosage is greater than 1 μg/patient and usuallygreater than 10 μg/patient if adjuvant is also administered, and greaterthan 10 μg/patient and usually greater than 100 μg/patient in theabsence of adjuvant. A typical regimen consists of an immunizationfollowed by booster administration at time intervals, such as 6 weekintervals. Another regimen consists of an immunization followed bybooster injections 1, 2, and 12 months later. Another regimen entails anadministration every two months for a prolonged period in excess of 12months. Alternatively, booster injections can be on an irregular basisas indicated by monitoring of immune response.

In certain embodiments, multiple doses are given over a period of time,each using a different immunogenic oligonucleotide in an appropriateamount, as indicated above.

The glycopolypeptides of the invention can also be used to detect aneutralizing antibody in a patient sample (e.g., a serum sample). Thismethod includes providing a glycopolypeptide of the invention,contacting the glycopolypeptide with a sample from an individual; anddetecting whether the glycopolypeptide binds specifically to an antibodypresent in the sample, wherein the detection of the antibody is carriedout using a label.

Exemplary labels include, without limitation, a radiolabel, fluorescentlabel, enzymatic label, chemiluminescent marker, biotinyl group, anepitope recognized by a secondary reporter, a magnetic agent, or atoxin.

The detection step is preferably carried using a suitable assay format.Exemplary assays include, without limitation, ELISA, radioimmunoassay,gel-diffusion precipitation reaction assay, immunodiffusion assay,agglutination assay, fluorescent immunoassay, immunoelectrophoresisassay, surface plasmon resonance assay, or biolayer interferometryassay. In certainly of these assay formats, a secondary antibody is usedto label the antibody bound specifically to the glycopolypeptide.Depending on the type of assay, the glycopolypeptide can be in thesolution phase or coupled to a solid surface.

EXAMPLES

The following examples are intended to illustrate practice of theinvention, and are not intended to limit the scope of the claimedinvention.

Materials and Methods for Examples 1-4

Proteins and ribosomes for PURE system translation: Hexa-histidinetagged IF1, IF2, IF3, EF-Tu, EF-G, EF-Ts, RF1, RF3, RRF, MTF, MetRS,GluRS, PheRS, AspRS, SerRS, ThrRS, ArgRS, GlnRS, IleRS, LeuRS, TrpRS,AsnRS, HisRS, TyrRS, ValRS, ProRS, AlaRS, CysRS, LysRS, and GlyRS wereexpressed in Escherichia coli BL21 Star (DE3) (Invitrogen) and purifiedas previously described (Shimizu et al., Nat Biotech 19:751-755 (2001);Josephson et al., J. Am. Chem. Soc. 127:11727-11735 (2005); Shimizu etal., Methods Mol Biol. Vol. 607, p 11-21 (2010); and Ma et al., RibosomeDisplay and Related Technologies; Douthwaite, J. A., Jackson, R. H.,Eds.; Springer New York: Methods Mol Biol. Vol. 805, p 367-390 (2012),which are hereby incorporated by reference in their entirety). Ribosomeswere prepared combining the previously described protocols (Shimizu etal., Methods Mol Biol. Vol. 607, p 11-21 (2010); Subtelny et al., J. Am.Chem. Soc. 130:6131-6136 (2008); and Ohashi et al., Biochem. Biophys.Res. Commun. 352:270-276 (2007), which are hereby incorporated byreference in their entirety) with some modifications. E. coli A19 wasgrown and harvested as previously described (Subtelny et al., J. Am.Chem. Soc. 130:6131-6136 (2008), which is hereby incorporated byreference in its entirety). The pelleted cells were washed with ˜300 mLof suspension buffer (10 mM HEPES-KOH, pH 7.6, 10 mM magnesium acetate,50 mM KCl, 7 mM β-mercaptoethanol) and spun at 5,000 g for 15 min. Thepelleted cells were lysed in suspension buffer using a bead-beater andthe cleared lysate was obtained by centrifuge (Subtelny et al., J. Am.Chem. Soc. 130:6131-6136 (2008), which is hereby incorporated byreference in its entirety). The supernatant (˜20 mL) was mixed with thesame volume of suspension buffer containing 3 M (NH₄)₂SO₄ andcentrifuged at 36,000 g for 30 min. The resulted supernatant wasfiltered through a 0.45 μm membrane and subjected to FPLC purificationto yield ribosomes as previously described (Shimizu et al., Methods MolBiol. Vol. 607, p 11-21 (2010) and Ohashi et al., Biochem. Biophys. Res.Commun. 352:270-276 (2007), which are hereby incorporated by referencein their entirety).

PURE System Translation:

The PURE translation system with homopropargylglycine instead ofmethionine was prepared as previously described (Shimizu et al., NatBiotech 19:751-755 (2001); Josephson et al., J. Am. Chem. Soc.127:11727-11735 (2005); Guillen et al., J. Am. Chem. Soc.134:10469-10477 (2012); Shimizu et al., Methods Mol Biol. Vol. 607, p11-21 (2010); Ma et al., Ribosome Display and Related Technologies;Douthwaite & Jackson, Eds.; Springer New York: Methods Mol Biol. Vol.805, p 367-390 (2012), which are hereby incorporated by reference intheir entirety) with slight modifications. The reaction contained 50 mMHEPES-KOH (pH 7.6), 12 mM magnesium acetate, 2 mM spermidine, 100 mMpotassium glutamate, 1 mM dithiothreitol (DTT), 1× Complete ULTRA,EDTA-free (Roche), 1 mM ATP, 1 mM GTP, 20 mM creatine phosphate(Calbiochem), 0.01 mg/l 10-formyl-5,6,7,8-tetrahydrofolic acid, 0.04ABS₂₈₀ creatine kinase (Roche), 0.85 units/mL nucleoside 5′-diphosphatekinase from bovine liver (Sigma), 6.8 units/mL myokinase from rabbitmuscle (Sigma), 100 units/mL inorganic pyrophosphatase, 48 ABS₂₆₀ tRNAfrom E. coli MRE 600 (Roche), 20 μg/mL MTF, 10 μg/mL IF1, 40 μg/mL IF2,10 μg/mL IF3, 10 μg/mL EF-Tu, 50 μg/mL EF-Ts, 50 μg/mL EF-G, 10 μg/mLRF1, 10 μg/mL RF3, 10 μg/mL RRF, 0.66 μM MetRS, 0.23 μM GluRS, 0.027 μMPheRS, 0.21 μM AspRS, 0.45 μM SerRS, 0.011 μM ThrRS, 0.021 μM ArgRS,0.27 μM GlnRS, 0.11 μM IleRS, 0.093 μM LeuRS, 0.23 μM TrpRS, 0.094 μMAsnRS, 0.21 μM HisRS, 0.18 μM TyrRS, 0.089 μM ValRS, 0.031 μM ProRS,0.070 μM AlaRS, 0.41 μM CysRS, 0.18 μM LysRS, 0.024 μM GlyRS, 1.2 μMribosomes, a mixture of 17 natural amino acids (3 mM each), withmethionine-, cysteine-, and histidine-omitted and pre-adjusted pH to 7.6with KOH, and 3 mM L-homopropargylglycine (Chiralix). To label thepeptide radioisotopically, the reactions also contained L-[³⁵S]-cysteine(Perkin Elmer) or [2,5-³H]-L-histidine (Moravek Biochemicals) inconcentrations totaling 0.002-3 mM together with non-radioactivecysteine/histidine. These reactions were assembled on ice and initiatedby the addition of mRNA (0.5-1.0 μM), followed by incubation at 37° C.for 1 h for mRNA display or 2 h for individual free peptide translation.

Click Reaction:

This optimized procedure was used in rounds 2-10 of selection, and inpreparation of individual peptides for binding studies. Man₉-azide wassynthesized as previously described (Temme et al., Chem. Eur. J.19:17291-17295 (2013), which is hereby incorporated by reference in itsentirety). The click reaction was performed combining the previouslydescribed protocols (Temme et al., Chem. Eur. J. 19:17291-17295 (2013)and Hong et al., Angew. Chem. Int. Ed. 48:9879-9883 (2009), which arehereby incorporated by reference in their entirety) with somemodifications. The dry pellets of peptides or fusions in 0.5 mLmicrocentrifuge tubes were redissolved in 2.5 μL of 200 mM HEPES-KOH (pH7.6) and 10 mM aminoguanidine hemisulfate (mixture A). In the case offusions, ˜0.05% (v/v) Triton X-100 was also added to the solution. 2.5μL of a freshly-prepared solution of 2 mM CuSO₄, 2 mMTris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand and 6 mMMan₉-azide was transferred to a capless 0.5 mL microcentrifuge tube(mixture B). 3 μL of freshly-prepared solution of 2.5 mM Man₉-azide and0.83 mM THPTA was added to a capless 0.5 mL microcentrifuge tube(mixture C). Sodium-L-ascorbate (less than 10 mg) was transferred to acapless 0.5 mL microcentrifuge tube. Then, the microcentrifuge tubescontaining mixtures A, B and C, and sodium-L-ascorbate were purged underargon flow in the following manner. The microcentrifuge tubes werecarefully positioned at the bottom of a 25 mL two-neck pear(pointy-bottom) flask. Positive argon pressure was applied through oneneck, while a rubber septum with a purge needle was used to vent thesystem from the other neck. After 1 h of efflux, the septum was removedand, under Ar efflux, a pipette was inserted into the flask to addmixture B to mixture A. The sodium ascorbate was dissolved in degassedH₂O to a final concentration of 100 mM, and 0.5 μL was added to the tubecontaining mixtures A and B. After recapping followed by 15 min of Arpurge, the vent needle was removed to keep the system under positivepressure. After an additional 1 h and 15 min, mixture C and anadditional 0.25 μL of 100 mM sodium ascorbate was added to the reaction.After recapping and another 15 min of Ar purge, the vent needle wasremoved. After an additional 75 min, the click reaction mixture wastaken out from the flask and quenched with 1.25 μL of 10 mM EDTA (pH8.0). At this point, the total reaction volume was reduced to ˜2-3 μLdue to evaporation.

mRNA Display Selection:

The libraries of glycopeptide-mRNA-DNA fusions were prepared bymodifying the previously described protocol to prepare the unnaturalpeptide-mRNA-DNA fusions with PURE system (Guillen et al., J. Am. Chem.Soc. 134:10469-10477 (2012), which is hereby incorporated by referencein its entirety). The fusions were radiolabeled with ³⁵S-cysteine(rounds 1 and 2) or ³H-histidine (rounds 3-10), and the yields invarious purification steps were monitored by liquid scintillationcounting. During the procedure, the integrity of the fusion formationwas checked (FIGS. 3A-C) by SDS-PAGE with visualization byautoradiography (in rounds 1 and 2) or fluorography (in rounds 3-10).Below, divided into subsections, the procedure in selection round 1 isfirst described, and then the modifications used in rounds 2-10 aredescribed.

Preparation of Puromycin-Linked mRNA in Round 1:

The puromycin-linked mRNA for selection round 1 was prepared as follows.The antisense strands of synthetic library DNA (the Fixed library:5′-CTAGCTACCTATAGCCGGTGGTGATGGTGGTGATGACCCAGAGAACCGGAGCCN₃₀CATN₃₀CATN₃₀CATTTAGCTGTCCTCCTTACTAAAGTTAACCCTATAGTGAGTCGTATTA-3′(SEQ ID NO: 17) and the Variable library:5′-CTAGCTACCTATAGCCGGTGGTGATGGTGATGGTGGCCTAAGCTACCGGAGCC(SNn)₃₂CATTTAGCTGTCCTCCTTACTAAAGTTAACCCTATAGTGAGT CGTATTA (SEQ ID NO: 18), whereuppercase N is an equimolecular mixture of G, A, T and C; S is anequimolecular mixture of G or C; lowercase n is a mixture of 40% T, 20%A, 20% G, and 20% C) were purchased from W.M. Keck BiotechnologyResource Laboratory, Yale University. The regions involved in the openreading frame in the constant regions of the two libraries were designedto have identical amino acid sequence but were not identical innucleotide usage, so that the libraries would be PCR-amplified withdifferent primer sets.

The Fixed and Variable library DNAs purified by denaturingpolyacrylamide gel electrophoresis (PAGE), 780 and 300 pmol,respectively, were transcribed in the presence of 1.2 eq. of the DNAcontaining T7 promotor sequence (5′-TAATACGACTCACTATAGGGTTA ACTTTAG-3′)(SEQ ID NO: 19) using MEGAshortscript kit (Ambion). The transcripts werepurified by denaturing 5% PAGE and photo-crosslinked withpuromycin-containing oligonucleotide XL-PSO, XuagccggugA₁₅ZZACCP, whereX is C6 psoralen, lowercase nucleotides have 2′OMe, uppercase A and Care DNA, Z is Spacer 9 and P is puromycin (W.M. Keck BiotechnologyResource Laboratory, Yale University), by 365 nm UV irradiation aspreviously described (Kurz et al., Nucleic Acids Res. 28:e83 (2000) andSeelig, B. Nat. Protocols 6:540-552 (2011), which are herebyincorporated by reference in their entirety).

Translation to form alkynyl peptide-mRNA fusions in round 1: Theradiolabeled alkynyl peptide-mRNA fusions for round 1 selection wereproduced as follows. The peptide-mRNA fusions were translated from 1 μMRNA, of which ˜50% was crosslinked with the puromycin-containingoligonucleotide XL-PSO, in 5.2 mL PURE system translation reactionscontaining [³⁵S]-cysteine for 1 h at 37° C. Following translation, KCland magnesium acetate was added to facilitate fusion formation (Liu etal., RNA-Ligand Interactions, Part B; Academic Press Inc: San Diego,Methods Enzymol. Vol. 318, p 268-293 (2000), which is herebyincorporated by reference in its entirety), incubated for a 15 min atroom temperature, and frozen as previously described (Josephson et al.,J. Am. Chem. Soc. 127:11727-11735 (2005) and Guillen et al., J. Am.Chem. Soc. 134:10469-10477 (2012), which are hereby incorporated byreference in their entirety).

Purification and cDNA Synthesis of Fusions in Round 1:

The mRNA-peptide fusions were captured on oligo(dT) cellulose (Ambion),washed as previously described (Seelig, B. Nat. Protocols 6:540-552(2011), which is hereby incorporated by reference in its entirety), andeluted with 0.1% (v/v) Tween-20 followed by 0.22 μm-filtration andethanol precipitation. The recovered library fusions were purified withNi-NTA agarose (Qiagen) under a denaturing condition to remove mRNA notfused with peptide using a similar procedure as previously described(Guillen et al., J. Am. Chem. Soc. 134:10469-10477 (2012), which ishereby incorporated by reference in its entirety), and desalted by gelfiltration using NAP-5 columns (GE Healthcare) with the gel filtrationbuffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 5 mMβ-mercaptoethanol, 0.2% (v/v) Triton X-100) according to themanufacturer's protocol. The fusions were pelleted by ethanolprecipitation and cDNA was synthesized using Superscript III ReverseTranscriptase (Invitrogen) with RT primers (5′-T₁₅GTGATGGTGGTGATGACCCAGAG-3′ (SEQ ID NO: 20) for the Fixed library,5′-T₁₅GTGATGGTGATGGTGGCCTAAGC-3′ (SEQ ID NO: 21) for the Variablelibrary) in the presence of Superase-In (Ambion) and 0.1% (v/v) TritonX-100 according to the manufacturer's protocol. The reverse transcribedfusions were pelleted by ethanol precipitation.

Click Glycosylation of Fusions in Round 1:

In round 1, the click reaction of fusions was not yet optimized and hadto be done twice to give the desired glycosylation efficiency. The firstclick reaction was done under Ar with a setting as described in thesection of “Click reaction” but with slightly different conditions: Thestarting volume was ˜6 times larger, THPTA concentration was twice, andthe addition of THPTA, Man₉-azide and sodium ascorbate in the middle ofthe reaction was not carried out. Since some insoluble pellets wereobserved at this point, the pellets were collected after click reactionby centrifugation and purified with Ni-NTA agarose under denaturingcondition as described above. The eluted fusions were combined with thesaved soluble fractions and desalted by gel filtration and ethanolprecipitation. The recovered fusions were re-subjected to glycosylationusing a condition similar to the optimized protocol as described in thesection entitled “Click reaction” and then ethanol-precipitated.

Selection in Round 1:

The pellets of glycosylated peptide-mRNA-cDNA fusions were redissolvedin 500 μL of selection buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1%v/v Triton X-100). The Fixed and Variable library fusions (yields of23.0 and 14.4 pmol, equivalent to 1.4×1013 and 0.86×1013 sequences,respectively) were individually incubated with 100 nM 2G12 (PolymunScientific) in 5000 μL of selection buffer at room temperature. 100 μLof 30 mg/mL Dynabeads Protein G magnetic beads (Invitrogen) in selectionbuffer was added to the mixture and kept suspended by tumbling for 20min at room temperature to capture complexes. The beads weremagnetically isolated and washed with 3×500 μL of selection buffer. Toelute the 2G12-binding fusions, the beads were resuspended in 100 μL ofselection buffer, heated at 70° C. for 30 min, chilled on ice for 5 minand incubated at room temperature for 10 min with tumbling. Thesupernatant was recovered and the beads were rinsed with 2×100 μL ofselection buffer. These solutions were combined as an eluted fraction.

PCR Amplification of cDNA of Selected Fusions in Round:

The cDNAs of eluted fractions were amplified by PCR using Taq DNApolymerase (Roche) with the forward primer (Library FP15′-TAATACGACTCACTATAGGGTTAACTTTAGTAAGGAGG-3′, SEQ ID NO: 22) and thereverse primer (5′-CTAGCTACCTATAGCCGGTGGTGATGGTGGTGATG ACCCAGAG-3′, SEQID NO: 23 for the Fixed library); 5′-CTAGCTACCTATAGCCGGTGGTGATGGTGATGGTGGCCTAAGC-3′, SEQ ID NO: 24 for the Variable library). Theamplified DNAs were purified by phenol extraction and ethanolprecipitation, and used for the transcription of the next selectionround.

Modification of the Procedures in Rounds 2-10 and Sequencing.

The fusion preparation and purification procedures were repeated for 10rounds except for the following changes. In rounds 3-10, the transcriptswere purified using MEGAclear kit (Ambion) and crosslinked with XL-PSO.The puromycin-modified RNA was then purified with denaturing PAGE withthe visualization of Gel Indicator RNA Staining Solution (BiodynamicsLaboratory) and 0.5 μM was used in translation reaction in the presenceof ³H-histidine. In rounds 2-10, the translation volume was reduced to0.22-0.52 mL and purifications and following procedures were scaledaccordingly. Ni-NTA agarose affinity purification was done only onceafter oligo(dT) cellulose purification, except in round 2, in whichfusions after reverse transcription were re-purified with Ni-NTA agaroseand desalted as described above. In round 2 and all subsequent rounds,the click reactions were done only once in the same or similarconditions as described in the “click reaction” section. In theselection parts, the essential differences of the conditions betweenselection rounds were as summarized in FIG. 5A. In the rounds withDynabeads Protein A (Invitrogen), bead amounts were twice as much asprotein G beads, because the 2G12-capturing capacity of Dynabeadsprotein A was lower than that of Protein G. In the rounds with 100 mMmannose, binding reactions and the first two wash steps were withmannose, but not in the third wash and elution steps. In rounds 4 and 6,the unglycosylated library was negatively selected for binding to 100 nM2G12 with protein G magnetic beads in the absence of mannose, to removeglycan-independent binders. In rounds 7-10, the negative selections weredone in the presence of 100 mM mannose prior to the positive selections,to remove glycopeptides that bind to protein A or G magnetic beadbinders. For sequencing after the selection in rounds 7 and 10, thePCR-amplified DNA was cloned into pCR2.1-TOPO vector (Life Technologies)without colony color selection to avoid unintentional biases.

Nuclease-Digestion of Library Fusions:

To monitor the number of glycans on the peptides in the fusions in everyselection round, a part of the cDNA-RNA-glycopeptide fusions (0.05-1pmol) was removed after the click reaction and desalted by ethanolprecipitation in the presence of linear acrylamide carrier (Ambion). Therecovered fusions were diluted in 6-7 μL of 200 mM ammonium acetate (pH5.3) with 1 unit of nuclease P₁ (Sigma), and incubated at 37° C. for 1 hto digest nucleic acids. Then, the solutions were neutralized with Trisbuffer and analyzed by SDS-PAGE.

Preparation of Individual Peptides and Glycopeptides:

To generate peptides from individual clones, the plasmids were used astemplates for PCR with primer sets (library FP1 and5′-CTAGCTACCTATTTGTCATCGTCGTCTTTATAATCCCGGTGGTGATGGTGGTGA TGACCCAG-3′,SEQ ID NO: 25 for the Fixed library members or CTAGCTACCTATTTGTCATCGTCGTCTTTATAATCCCGGTGGTGATGGTGATGGTGGCCTAA-3′, SEQ ID NO: 26 for theVariable library members) and the PCR products were used for T7transcription. The resulting mRNAs were purified by denaturing PAGE orMEGAclear kit (Ambion), and 1 μM RNA was used in PURE system translation(reaction volume of translation varied). Typically, 25 μL of translatedreaction was diluted with 100 μL of binding buffer (50 mM Tris-HCl, pH7.8, 300 mM NaCl, 5 mM β-mercaptoethanol) and 25 μL of Ni-NTA agarosesuspension (Qiagen), and tumbled at room temperature for 1 h. The resinswere transferred to 0.22 μm spin-filter rinsing with 100 μL of bindbuffer, and washed with 3×200 μL of bind buffer and 2×200 μL of washbuffer (50 mM Tris-HCl, pH 7.8, 5 mM β-mercaptoethanol). The boundpeptides were eluted with 2×25 μL of 0.1% TFA. The eluted peptides wereanalyzed by MALDI-TOF MS, using α-Cyano-4-hydroxycinnamic acid matrix(Sigma), with or without desalting with ZipTip C₁₈ resin (Millipore).For calibration of MALDI-TOF-MS, at least two of the followingstandards, bovine insulin, E. coli thioredoxin and/or horse apomyoglobinwere used. To quantitate peptide yields, the radioactivities weremeasured by liquid scintillation counting. For the click reaction, thetranslated and purified peptides were mixed with 0.1% (v/v) TritonX-100, and then dialyzed against H₂O containing 0.1% (v/v) Triton X-100using Slide-A-Lyzer MINI Dialysis Devices, 3.5K MWCO (Thermo Scientific)overnight to desalt. After dialysis, the peptides were divided into twoportions: one was glycosylated via the click reaction, while the otherwas saved as a non-glycosylated peptide control. The peptides to beglycosylated (typically less than 5 pmol) were evaporated by speedvac ina 0.5 mL microcentrifuge tube for use in click glycosylation. Since theefficiency of the click reaction was not always high with round 10winners, crude glycosylated peptides were subjected to 2G12 affinitypurification to obtain the highest-clicked fraction, as follows. Theglycosylated peptide (<4 pmol) was incubated with 100 nM 2G12 inselection buffer (40 μL) at room temp. The solution was then tumbled 30min with 0.12 mg of equilibrated Dynabeads Protein G to capture2G12-glycopeptide complex. Beads were then washed with 3×40 μL ofselection buffer and resuspended in 10 μL of selection buffer. Theresuspended beads were heated at 70° C. for 30 min to denature 2G12 andelute glycopeptides, chilled on ice for 5 min, and tumbled at roomtemperature for 10 min. The magnetically isolated supernatant wasrecovered and the beads were rinsed with 10 μL of selection buffer. Thesupernatant and the rinsed solution were combined as the purifiedglycopeptide fraction, and the yields were measured by liquidscintillation counting (the recovery of radioactivity was typically in arange of 25-55% of input radioactivity).

SDS-PAGE of Nuclease-Digested Fusions and Glycopeptides:

Unless otherwise noted, SDS-PAGE of nuclease-digested fusions andglycopeptides was done as follows. A 4-20% gradient precast gel(Bio-Rad) was run using a rapid protocol (300 V for 16-20 min).Precision Plus Protein Dual Xtra Standards (Bio-Rad) were used as amolecular weight marker. To visualize the ³⁵S-labeled peptides byautoradiography, gels were soaked in fixing solution (22.5% acetic acidand 5% ethanol) with shaking for 15 min, dried on filter paper, andexposed to a phosphorimager screen to analyze using Storm Phosphorimager(Amersham). To visualize the ³⁵H-labeled peptides by fluorography, gelswere treated with NAMP100 Amplify Fluorographic Reagent (GE Healthcare)according to the manufacture's protocol, then dried and exposed to X-rayfilms at −80° C.

Binding Curve and K_(D) Determination of 2G12-Glycopeptide Interaction:

For round 10 winners, 0.12-0.2 nM radioactive glycopeptides wereincubated with 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32 or 64 nM 2G12 in 40 μLof selection buffer at room temperature for 1 h. Then, the solution wasadded to 0.12 mg of pre-equilibrated Dynabeads Protein G and tumbled atroom temperature for 30 min. The supernatant was removed and the beadswere washed with 3×40 μL of selection buffer. The radioactivities of thesupernatant and wash solutions were measured by liquid scintillationcounting as unbound fractions. Since the direct usage of the capturedglycopeptides on the beads in liquid scintillation counting partiallysuppressed the radioactivity detection in the case of ³H-label, thebound glycopeptides were eluted and separated from the beads in afollowing manner. The beads were resuspended in 40 μL of selectionbuffer, heated at 70° C. for 30 min to elute the bound glycopeptides,chilled on ice for 5 min, and tumbled at room temperature for 10 min.The supernatant was removed, and the beads were washed with 40 μL ofselection buffer and resuspended in 40 μL of selection buffer. Theradioactivities of these solutions and suspensions were measured byliquid scintillation counting separately and the values were combined asapparent bound fractions. The measured radioactivity of the fractionwhich bound to the beads without 2G12 (ranging from 0-6% of the totalradioactivity in the assay) was subtracted as background from theradioactivity bound to the beads with 2G12, and the difference wasdivided by the total radioactivity to determine the percentages bound to2G12. For glycosylated and non-glycosylated 7V8, the same procedure wasdone except for the following changes: the volume of each solution wasreduced to 30 μL, 2 nM radioactive glycopeptide was incubated with 0,3.125, 6.25, 12.5, 25, 50, or 100 nM 2G12, and 0.18 mg Dynabeads ProteinG was used to capture 2G12. All experiments were done at least intriplicate. K_(D)s were calculated as described in the footnote of Table4 (below).

Analysis of Competition of Glycopeptides and gp120 or Mannose for2G12-Binding and Non-Glycosylated Peptide Binding to 2G12 of Round 10Winners:

The procedure was essentially same as described in the previous sectionwith slight modification as follows. The volume of binding reaction was20-30 μL and other volumes were also adjusted accordingly. 200 nM 2G12in selection buffer was pre-mixed with or without 400 nM 6×His-taggedgp120(JRFL)(HIV-1) (Immune Technology) or 1 M mannose, and further mixedwith the same volume of 0.4 nM radioactive glycopeptides ornon-glycosylated peptides for binding reaction. The solutions wereincubated at 37° C. for 30 min to equilibrate binding competition andthen incubated at room temperature for 30 min to stabilize thecomplexes. Pre-equilibrated protein G magnetic beads were added to givea final concentration of 6 mg/mL. The separation of unbound fractionsand bound fractions was done as described above, except that 0.5 Mmannose was added to the washing solution in the case of mannosecompetition. All experiments were done 3× or more.

Preparation of Synthetic Peptide 10F2 (1):

The unglycosylated peptide 10F2,fXHPYNTSRTSAXXAALKXQVTDXYALALFHRIL-GSGSGC(StBu)A, SEQ ID NO: 60 wheref=formyl and X=homopropargylglycine, was prepared by Fmoc solid-phasepeptide synthesis using Pentelute's recent rapid flow-based method(Simon et al., ChemBioChem Early View DOI: 10.1002/cbic.201300796 (2014)which is hereby incorporated by reference in its entirety). 76 mg (25μmol scale) of trityl chemmatrix resin, loaded with 0.33 meq/g alanineby standard procedures (Chan et al., Fmoc Solid Phase Peptide Synthesis;Oxford University Press: Oxford, UK (2000), which is hereby incorporatedby reference in its entirety), was subjected to 39 cycles of peptidecoupling and Fmoc deprotection, with thermal heating to 60° C. (seeTable 1 for detailed conditions).

Cysteine and histidine couplings were performed with a lower baseconcentration to avoid racemization and homopropargylglycine couplingswere performed as batch reactions to conserve amino acid. AfterN-terminal formylation p-nitrophenyl formate, the peptide was cleavedand deprotected using cleavage cocktail B (87.5/5/5/2.5TFA/water/Phenol/iPr₃SiH), and the peptide was triturated four timeswith cold ether to afford 38 mg of crude solid. 5 mg of this wasredissolved in 200 μL DMSO, diluted with 200 μL water, and purified byRP HPLC (Waters Symmetry 300 C4, 5 μm, 10×250 mm, 4 mL/min, 2-42% MeCNin H₂O w/0.1% Formic Acid, over 60 min, retention time 52 min) to afford1.5 mg of product, corresponding to an overall SPPS yield of 11% if thewhole batch had been purified. LR ESI-MS: obs. average base peaks 868.79[M+5H]⁵⁺, 1085.75 [M+4H]¹⁺, 1447.23 [M+3H]³⁺, corresponding to 4338.9obs. average mass, calc. average mass 4339.9.

TABLE 1 Peptide Synthesis Detailed Conditions. Cou- AA/ pling Couplingmmol Base Coupling Coupling Reagent Conc. AA Conc. Flow Rate Time MostAA* HATU 0.33M 1 0.95M 6 ml/min 30 sec Cys(StBu) HATU 0.33M 0.15 0.86MN/A 10 min His(Trt) HATU 0.33M 1 0.29M 6 ml/min 30 sec HPG HATU  0.3M0.15 0.86M N/A 10 min Gly HBTU 0.33M 1 0.95M 6 ml/min

The general coupling procedure follows Pentulute's procedure (Simon etal., ChemBioChem Early View DOI:10.1002/cbic.201300796 (2014), which ishereby incorporated by reference in its entirety) with modifications.After coupling and after Fmoc deprotection, the resin was washed with 20ml of DMF at a flow rate of 10 ml/min. Fmoc deprotection was carried outat flow rate of 10 ml/min. Fmoc deprotection solution was 20% piperidinein DMF up until the coupling of aspartic acid, after which a solution of19% piperidine/1% formic acid in DMF was used to prevent aspartimideformation.

Fmoc-Cys(StBu)-OH and Fmoc-HPG-OH were coupled outside of the reactor.Swelled resin was transferred to a 15 mL conical tube with a stir bar.0.15 mmol amino acid and 0.15 mmol HATU were dissolved in 425 μL DMF,and 75 μL DIPEA was added just before adding to resin. The couplingreaction was allowed to take place under nitrogen for 10 minutes at 60°C. with stirring. After the reaction, the resin was transferred to thereactor for washing and Fmoc deprotection.

After peptide synthesis was complete, the N-terminus was formylated. Theswelled resin was transferred to a 15 ml conical tube with a stir bar.0.25 mmol 4-nitrophenylformate was dissolved in 632 μl DMF (0.33 Mfinal). 125 μL DIPEA (0.86 M final) was added just before addition.Formylation was allowed to occur at 60° C. for 8 minutes while stirringunder nitrogen. Next, the supernatant was removed, fresh reagents wereadded, and formylation was repeated. This was done again for a total of3-8 minute periods. After the reaction, the resin was washed with DMF5×10 ml and DCM 3×10 ml.

The peptide was cleaved from the resin with 10 ml of a cleavage cocktailB containing 87.5/5/5/2.5 TFA/phenol/water/TIPS. The resin and cocktailwere tumbled at room temperature for 90 minutes. The resin was filteredand washed 3×4 ml DCM. The filtrate was concentrated by rotaryevaporation and transferred to a 15 ml conical tube. The peptide wastriturated with 5×10 ml cold ether to give 35 mg crude peptide.

4.5 mg of crude peptide was purified by HPLC on a Waters Symmetry300 C4column (4.6×250 mm, 5 μm particle size) following a 98% A/2% B to 58%A/42% B gradient over 60 minutes with a flow rate of 4 ml/min, wheresolvent A is water/0.1% formic acid and solvent B is acetonitrile/0.1%formic acid.

Glycosylation of Synthetic Peptide 10F2:

10F2 peptide (0.6 mg, 0.14 μmol, 1 equiv.) and Man₉-azide (1.5 mg, 0.97μmol, 7.0 equiv.) were combined in a 0.5 mL Eppendorf tube byevaporation of stock solutions (tube A). A second tube was prepared,containing 9.8 μL (0.98 μmol, 3.0 equiv.) of a 100 mM solution of THPTAligand and 9.0 μL (0.90 μmol, 2.8 equiv.) of a 100 mM solution of CuSO₄(tube B), and the tube was evaporated to dryness. Sodium ascorbate (3.0mg, 15.2 μmol, 47 equiv.) was placed in a third tube (tube C). The threetubes were placed in a 2-neck pear (pointy-bottom) flask, and nitrogenatmosphere was established by cycles of vacuum and nitrogen refill.Under nitrogen efflux, 150 μL DMSO (degassed by freeze-pump-thaw) wasadded to dissolve the peptide and sugar in tube A, and 75 μL H₂O(degassed by freeze-pump-thaw) was added to dissolve the contents ofeach of tubes B and C. The contents of tube B, and then tube C, weretransferred by syringe to tube A. The resulting homogenous mixture wasallowed to react under nitrogen atmosphere for 20 hours, at which timeUPLC/MS analysis showed nearly complete conversion. The reaction wasquenched by addition of TMEDA (1.5 μL, 3.22 μmol, 10 equiv.) andconcentrated in vacuo. The residue was purified by RP-HPLC (same columnand gradient method as for the unglycosylated 10F2 peptide, retentiontime 45 min) to afford pure glycopeptide 2. ESI-HRMS: obs. base peaks:2058.0088 [M+6H]⁶⁺, 2469.4028 [M+5H]⁵⁺, 3086.7759 [M+4H]¹⁺, deconvolutedmass 12334.962, calc. 12334.980±0.128.

Biotinylation of 10F2 Glycopeptide and Determination of 2G12 Binding byBLI (BioLayer Interferometry):

200 μg 10F2 glycopeptide in 5.5 μL water was treated with 6.5 mL of 50mM TCEP.HCl/1M Tris-HCl buffer, pH 7.8, under argon, using the sameinert gas setup employed in the click procedure. After 4.5 h, thereaction mixture was injected into HPLC (Waters Symmetry, 300 C4, 5 μm,4.6×250 mm, 1 mL/min, 2-42% over 60 min, retention time 46.8 min).

The 2G12 binding of the resulting biotinylated glycopeptide 3 wasdetermined using a BLItz instrument (Fortebio). Biotin-10F2 was loaded(120 sec) onto a streptavidin biosensor as a 500 nM solution in Buffer 1(20 mM Tris pH 7.5, 150 mM NaCl, 2 mM MgSO₄, 0.20 mg/mL BSA, 0.02%Tween-20). The sensor was washed with Buffer 1 for 60 sec, after whichtime the net response due to loading was observed as 0.2 nm. The sensorwas then equilibrated with Buffer 2 (20 mM Tris pH 7.5, 150 mM NaCl, 2mM MgSO₄, 2.0 mg/mL BSA, 0.1% v/v Tween-20) for 90 sec. 2G12 (preparedin Buffer 2) was associated at several concentrations (0.5, 1, 2, 4, 8,16, 32 nM, in random order) for 600 sec, followed by dissociation intoblank Buffer 2 for 600 sec. After each 2G12 dissociation, the sensor wasregenerated to remove remaining 2G12 by treatment with buffer 3 (10 mMglycine-HCl, pH 2.5) for 120 sec, followed by 60 sec of wash with Buffer1 and further washes to re-equilibrate the tip with Buffer 2. Throughoutthe experiment, the shake rate was set at 1800 rpm. The use of Buffer 2(with high BSA) was important during association/dissociation to preventnonspecific 2G12/streptavidin interactions, while Buffer 1 (low BSA) wasrequired during loading of the glycopeptide to the sensor surface. Tofurther correct for residual non-specific interactions, the data wasreferenced to a blank run using 0.5 nM 2G12 on a sensor containing noloaded peptide. The data was fit to a 1:1 binding model, yielding rateconstants of k_(on)=11.1±0.4×10⁴ M⁻¹ s⁻¹ and k_(off)=1.51±0.02×10⁻⁴ s⁻¹,corresponding to a K_(D) of 1.37±0.02 nM (Table 2).

TABLE 2 BLI Curve Fit Parameters Conc K_(D) ka ka kd kd Rmax (nM) (M)(M⁻¹s⁻¹) error (s⁻¹) error Rmax error Req 0.5 1.368e−9 1.106e5 3.989e21.513e−4 1.51e−6 1.797 0.2195 0.4810 1 1.368e−9 1.106e5 3.989e2 1.513e−41.51e−6 1.615 0.01391 0.6817 2 1.368e−9 1.106e5 3.989e2 1.513e−4 1.51e−61.662 0.01177 0.9866 4 1.368e−9 1.106e5 3.989e2 1.513e−4 1.51e−6 1.6910.01057 1.26 8 1.368e−9 1.106e5 3.989e2 1.513e−4 1.51e−6 1.601 0.0081361.367 16 1.368e−9 1.106e5 3.989e2 1.513e−4 1.51e−6 1.535 0.005597 1.41432 1.368e−9 1.106e5 3.989e2 1.513e−4 1.51e−6 1.453 0.003017 1.394

Example 1—Fixed and Variable Peptide Library Design for the DirectedEvolution of Glycopeptides

Two libraries of ˜33-mer peptides with glycosylation sites locatedeither in “Fixed” or “Variable” locations were employed. The Fixedlibrary (FIG. 2C, top) contains three potential Man₉-glycosylation sitesencoded by AUG codons at the “fixed” positions 1, 12, and 33, and eachsite is followed by 10 random amino acid residues (X1₀) encoded by NNNcodon, where N is an equimolecular mixture of G, A, U, and C, and inwhich AUG codon appears in a ratio of 1.6% per position. The Variablelibrary (FIG. 2C, bottom) contains the N-terminal Man₉-glycosylationsite encoded by the AUG start codon (due to the necessity of the AUGcodon for the translation start) followed by 32 random amino acidresidues (X₃₂). X is encoded by doped N′NS codons, where N′ is a mixtureof 40% A, 20% G, 20% U and 20% C, S is an equimolecular mixture of G andC, and in which AUG codon appear in a rate of 5% per position.

Example 2—Directed Evolution of Fixed and Variable Peptide LibrariesAgainst HIV Broadly Neutralizing Antibody 2G12

These libraries of ˜10¹³ sequences were subjected in parallel to 10rounds of selection for binding to 2G12. mRNA-displayed-glycopeptideswere incubated with successively lower concentrations of 2G12, and boundcomplexes were retrieved from solution alternately with Protein A orProtein G magnetic beads. Bound fusions were eluted by heating, in whichthe gp120-binding activity of 2G12 was selectively inactivated withoutharming the nucleic acid tags (FIGS. 4A-4D).

FIG. 5A shows the percent recovery of the library after each round ofselection, as monitored by scintillation counting of radioactive³⁵S-cysteine or 3H-histidine. Because the recovery after round 2 wasquite high (˜10%), the selection stringency was increased by adding 100mM mannose as a competitor in all subsequent rounds. Glycan-independentbinders were removed from the library in rounds 4 and 6 by counterselection of the library prior to the CuAAAC reaction. Counterselections without 2G12 (Protein A or protein G beads only) were usedstarting at round 7 to remove possible bead binders. Selection rounds1-7 were performed at room temperature, and rounds 8-10 were performedat 37° C., to remarkable effect (vide infra).

By the end of round 7, library binding to 100 nM 2G12 had grown to ahigh level. However, an undesired trend toward very high multivalencywas also observed throughout these room-temperature selection rounds.This can be seen by SDS-PAGE of the nuclease P₁-digested library justprior to each selection round (FIG. 5B, arrows), where separate bandsare visible for library species containing different numbers of glycans.This interpretation was confirmed by sequencing of round 7 clones (Table3 below), which showed that nearly all peptides contained 6-12glycosylation sites.

TABLE 3 Selected Clones from round 7. SEQ Potential ID Glyco. LibraryClone Sequence^(a) NO: Sites Fixed 7F1 XYYLSVYPSYSXYFSSSYVVWPXPGHRLLIGLE27  3 7F10 XXEHKLTXLPLXSTDIFLVLLXXFGTTITQVSL 28  6 7F6XYLPDWXLKSLXLSKWRLPEXFXSPFXLELHXS 29  7 7F8XLTNITLQXSRXHLLWLHXHDLXXDLCRIXLRS 30  7 7F5XVLTPTTKXXVXQSPXYFXRSNXLSKXYDYQRL 31  8 7F11XXIXNSXRIDVXXSNFVHAKSTXVGQRHXGGVG 32  8 7F12XSXTXQFSHFWXRHXWESXNRWXLARTXDTPID 33  8 7F16XXCHCLPSHYXXLRFCPXTGSVXDXGLKRXVYH 34  8 7F2XAKFDEXXAXLNXSRXSSYLXXLXTGRTWPH 35  9 7F15XTFEXLPRSDSXRXLTXPXXHRXYXIYRGYSNR 36  9 7F17XSYSXSPRDPNXXIKFLXSRTXXRNPXNVIGSX 37  9 Variable 7V12XHISTNCXPWRYWSIICXXPTWKTVHQXXKTKD 38  6 7V6XCSRKXACLSRANLXRXRSXXKRRXTXNTSFTX 39  9 7V8XIRXRTPTSRLXSTXRGXTXNXTSXITPRNDXI 40  9 7V10XTPFTXAYXTRRKPXXFPIXHRXKSRTPLXXGK 41  9 7V3XKXNXRIWNPXXNNWSXDTASXLRLXSWXLNXX 42 11 7V4XTSIXDNTXXLSVNXNRXKINRTLXXXXHXSTX 43 12 7V9XCXKXYAPNXYDLXPXRXHWXPNVLXPLXSXRX 44 12 ^(a)Only the sequence of therandom region (position 1-33) is shown. All peptide sequences used inthe 2G12-binding assay were followed by a linker, a His₆-tag and aFLAG-tag (GSGSLGHHHHHHRDYKDDDDK, SEQ ID NO: 1) for purification andradiolabeling purposes.

One of these, peptide 7V8 (SEQ ID NO: 40), exhibited a K_(D) of 17 nMfor binding to 2G12 (see Table 4). Although this 2G12 recognition istighter than that of most reported oligomannose clusters (Ni et al.,Bioconjugate Chem. 17:493-500 (2006); Joyce et al., Proc. Natl. Acad.Sci. U.S.A. 105:15684-15689 (2008); Astronomo et al., J. Virol.82:6359-6368 (2008); Astronomo et al., Chem. Biol. 17:357-370 (2010);Luallen et al., J. Virol. 82:6447-6457 (2008); Luallen et al., J. Virol.83:4861-4870 (2009); Agrawal-Gamse et al., J. Virol. 85:470-480 (2011);Ciobanu et al., Chem. Commun. 47:9321-9323 (2011); Marradi et al., J.Mol. Biol. 410:798-810 (2011); Li et al., Org. Biomol. Chem. 1:3507-3513(2003); Li et al., Org. Biomol. Chem. 2:483-488 (2004); Wang et al.,Chem. Biol. 11:127-134 (2004); Krauss et al., J. Am. Chem. Soc.129:11042-11044 (2007); Wang et al., Org. Biomol. Chem. 5:1529-1540(2007); Wang et al., Proc. Natl. Acad. Sci. US A. 105:3690-3695 (2008);Gorska et al., Angew. Chem. Int. Ed. 48:7695-7700 (2009); Doores et al.,Proc. Natl. Acad. Sci. U.S.A. 107:17107-17112 (2010); and Clark et al.,Chem. Biol. 19:254-263 (2012), which are hereby incorporated byreference in their entirety). 6-10 glycans is far more than the 3 or 4gp120 glycans thought to be involved in 2G12 binding (Scanlan et al., J.Virol. 76:7306-7321 (2002); Calarese et al., Science 300:2065-2071(2003); and Calarese et al. Proc. Natl. Acad. Sci. U.S.A.102:13372-13377 (2005), which are hereby incorporated by reference intheir entirety). Moreover, none of the sequences obtained werereplicates, indicating that the library had not yet converged to thebest possible sequences. For this reason, selection was continued foradditional rounds.

Given the high multivalency concerns, subsequent selection rounds werecarried out at 37° C. because of a striking temperature effect observedin related studies with SELMA selection of glycosylated DNA libraries(Temme et al., J. Am. Chem. Soc. 136:1726-1729 (2014), which is herebyincorporated by reference in its entirety). In that work, increasing thetemperature of the 2G12 selection step to 37° C. was found todramatically favor sequences with lower multivalency and much strongerbinding. After applying this modification to the next three rounds ofglycopeptide selection, a parallel trend in the results was observed:low-valent binders—barely visible in the whole-library gel at thebeginning of round 8—completely took over both libraries (FIG. 5B,boxes).

The selected round 10 glycopeptides are listed in Table 4, along withtheir measured binding affinity for 2G12.

TABLE 4 Binding Constants of Selected- and non-Selected Glycopeptides.Library SEQ Poten. (round ID Glyco. K_(D) Fmax obtained) CloneSequence^(a) NO: Sites  [nM]^(b) [%]^(c) Fixed  6EXQTACPSPAFLXLSRSAHYFHAXHPTSAAPDIS 45 3 >128 ND^(d) (before 1) Variable12G XYKNIPSTTXNLYSKPXATVTTLKCKLNGNRIS 46 3 >128 ND^(d) (before 1)Variable  7V8 XIRXRTPTSRLXSTXRGXTXNXTSXITPRNDXI 40 9   17 ± 108 ± (7)   5.6  12 Fixed 10F6 XLXFIRIYPTRXQYVYHAPLLTXVRXSPTGPLI 15 5    0.54 ± 87 ± (10)    0.043   1.3 10F2 XHPYNTSRTSAXXAALKXQVTDXYALALFHRIL 13 5   0.60 ±  86 ±    0.045   1.2 10F12 XCYVTVIPAXNXPEARLGIVCHXPGIRRGKALY16 4    0.77 ±  90 ±    0.084   2.0 10F5XSPHLPVLLCKXVLNDGRRIVQXSCELPXVRRS 14 4    0.97 ±  93 ±    0.13   2.710F8 XLLKXVDQSRLXPVPGIGVTLHXRSIPYSYLPI  4 4    2.6 ±  97 ±    0.23   2.210F3 XDTLHLKQIGGXPNCITQQDVRXTSIPYTYTWP  3 3    3.0 ± 100 ±    0.31   2.710F9 XRSTLNSLEYRXQYATEDPRIRXASIPYTYWWP  5 3    3.1 ±  86 ±    0.17   1.2Variable 10V1 XATKTNCKREKTXDNHVTIXRSIPWYTYRWLPN  6 3    1.9 ±  97 ± (10)   0.17   2.1 10V9 XTSIPYTYLNRSLWTNYRVNSWSXSKNVNVXPL 11 3    3.9 ±  85 ±   0.11   0.68 10V8 XVLPTIISTNVNPFRXLSIPTYTYLXPITWGEI 10 3    4.6 ± 94 ±    0.34   2.0 ^(a)Only the sequence of the random region (position1-33) is shown. All peptide sequences used in the 2G12-binding assaywere preceded by an N-terminal formyl group and followed by a linker, aHis₆-tag and a FLAG-tag (GSGSLGHHHHHHRDYKDDDDK, SEQ ID NO: 1) forpurification and radiolabeling purposes. “X” denotes potentialMan₉-glycosylation sites encoded by the AUG codon. ^(b,c)In the assay,the peptides were radiolabeled with either ³⁵S-cysteine or ³H-histidineas described above, and incubated with various concentrations of 2G12,and 2G12-peptide complexes were isolated with magnetic protein G beads.Percentages of the fractions bound were calculated from radioactivitymeasured by liquid scintillation counting as described above. K_(D) andF_(max) were calculated by fitting F_(bound) = (F_(max) [2G12])/(K_(D) +[2G12]) to average data points. Errors reported are the standard errorof the curve fit. ^(d)Not Determined.

Example 3—Identification of a Peptide Consensus Motif in Fixed andVariable Round-10 Selected Glycopeptides

Sequencing of 24 clones from each library (Tables 4, 5) confirmed thelow number of glycosylations (2 to 5) and revealed a high degree ofsequence convergence.

TABLE 5 Selected Clones from Round 10. SEQ Potential No. ID Glyco.clones Library Clone Sequences^(a) NO: Sites (in 24) Fixed 10F5XSPHLPVLLCKXVLNDGRRIVQXSCELPXVRRS 14 4  4/24 10F2XHPYNTSRTSAXXAALKXQVTDXYALALFHRIL 13 5  3/24 10F12XCYVTVIPAXNXPEARLGIVCHXPGIRRGKALY 16 4  2/24 10F6XLXFIRIYPTRXQYVYHAPLLTXVRXSPTGPLI 15 5  1/24 10F16XVRSAAVDTSPXTSSSQNAILLXFSYDVCLFDL 47 3  1/24 10F20XIALTSNCYLNXGPRIFRYDVGLTQLCQGRRRS 48 2  1/24 10F3XDTLHLKQIGGXPNCITQQDVRXTSIPYTYTWP  3 3  4/24 10F23XDTLHLKQIGVXPNCITQQDVRXTSIPYTYTWP 49 3  1/24 10F8XLLKXVDQSRLXPVPGIGVTLHXRSIPYSYLPI  4 4  4/24 10F9XRSTLNSLEYRXOYATEDPRIRXASIPYTYWWP  5 3  2/24 10F18XFSTANIYGAPXNTDXRLEHRQXKSIPYTYYWS 50 4  1/24 10F24XERPSLXCGLSXLTSGGTQSSVXRSIPFYTYWW 12 4  1/24 Var. 10V1XATKTNCKREKTXDNHVTIXRSIPWYTYRWLPN  6 3 14/24 10V14XATKTNCKREKTIDNHVTIXRSIPWYTYRWLPN 51 2  2/24 10V2XATKTNFKREKTXDNHVTIXRSIPWYTYRWLPN  7 3  1/24 10V6XATRTNCKREKTXDNHVTIXRSIPWYTYRWLPN  8 3  1/24 10V11XATKTSCKREKTXDNHVTIXRSIPWYTYRWLPN  9 3  1/24 10V9XTSIPYTYLNRSLWTNYRVNSWSXSKNVNVXPL 11 3  4/24 10V8XVLPTIISTNVNPFRXLSIPTYTYLXPITWGEI 10 3  1/24 ^(a)Consensus sequences arein bold. 10F23, 10V14, 10V2, 10V6 and 10V11 contain a mutated amino acidfrom their potential parent sequences, 10F3 and 10V1, respectively.

Many repeat sequences were observed, as was a peptide consensus motif,XxSIP(−/x)YTY(L/xW)(−/x)P, where X denotes Man9-HPG and x denotes avariable amino acid. This motif was present in some clones from bothlibraries, and apparently arose from convergent evolution in multiplesequence families, as it is located sometimes early, sometimes late inthe sequences. 10 glycopeptides were prepared without mRNA tags by invitro translation without the puromycin linker (FIGS. 6A-6B), andK_(D)'s were determined by incubation of the glycopeptide with variousconcentrations of 2G12, followed by capture on protein G beads andquantification of radioactivity. All 10 of the tested glycopeptides wererecognized tightly by 2G12, with K_(D)'s in the range of 0.5-5 nM,similar to the strength of 2G12-gp120 interaction (Table 4 and FIG. 7)(2G12-gp120 K_(D)=5.8 nM) (Hoorelbeke et al., J. FEBS Lett. 587:860-866(2013), which is hereby incorporated by reference in its entirety). Someof these peptides lacked the peptide consensus motif, and all weredependent on glycosylation for 2G12 binding (FIG. 8A), indicating thatthe glycans are the major element recognized by the antibody. This wasfurther confirmed by studies showing a significant reduction in bindingwhen either 0.5M mannose. Moreover a reduction in binding observed with200-800 nM recombinant gp120 (FIGS. 8A-B) added to the assay shows thatthe selected glycopeptides compete with gp120 for binding its site on2G12. In contrast to round 10 selected peptides, clones picked from thelibraries prior to selection showed very little binding to 2G12 atconcentrations up to 128 nM (Table 4, FIGS. 9A-C), indicating that notall peptide backbones are suitable for highly antigenic presentation ofthe carbohydrates.

Example 4—Confirmation of 2G12 Binding Affinity to Round-10 SelectedGlycopeptides Using BioLayer Interferometry

To confirm that the results presented in Table 4 were not artifacts ofribosomal translation, glycopeptide 10F2 was chemically synthesized andcharacterized. The 2G12 binding affinity of glycopeptide 10F2 wasconfirmed using an alternate assay, BioLayer Interferometry, (“BLI”)(Abdiche et al., J. Anal. Biochem. 377:209-217 (2008), which is herebyincorporated by reference in its entirety). Pentelute's new Rapid FlowSolid Phase Peptide Synthesis method (Simon et al., ChemBioChem EarlyView DOI: 10.1002/cbic.201300796 (2014), which is hereby incorporated byreference in its entirety) in which activated amino acids are flowedthrough a thermally-heated reactor containing peptide synthesis resin,was used to prepare the 10F2 peptide. In this manner alkyne-containingpeptide 1, in which the C-terminal His6 tag of the ribosomal peptide wasreplaced by an -StBu-protected cysteine, was obtained (FIG. 10A). CuAAACglycosylation proceeded to near completion and HPLC purificationafforded the desired glycopeptide 2, whose identity was confirmed bymass spectrometry. Reductive deprotection of the cysteine and immediatetrapping with a maleimide-biotin reagent, appended the biotin necessaryfor immobilization to the streptavidin biosensor surface used in the BLIassay. After immobilization of the biotinylated glycopeptide 3 on thesensor, 2G12 was associated to the surface at several concentrations,followed by dissociation in blank buffer (FIG. 10B). The resultingresponse curves were fit globally to a 1:1 binding model and affordedrate constants of k_(on)=11.1±0.4×10⁴ M⁻¹ s⁻¹ and k_(off)=1.51±0.02×10⁻⁴s⁻¹, corresponding to a K_(D) of 1.37±0.02 nM. This affinity measurementis in reasonable agreement with the measurement ofribosomally-translated 10F2 in the bead-based assay. Moreover, thisinteraction is both kinetically and thermodynamically comparable to thatmeasured for the 2G12-gp120 interaction (k_(on)=6.6×10⁴ M⁻¹ s⁻¹,k_(off)=3.8×10⁻⁴ s⁻¹, K_(D)=5.8 nM) (Hoorelbeke et al., J. FEBS Lett.587:860-866 (2013), which is hereby incorporated by reference in itsentirety).

The 2G12 recognition observed for the Man₉ glycopeptides obtained in thepreceding Examples represents an enhancement of up to ˜360,000-foldcompared with monovalent Man₉ glycan (K_(D)=180 μM) (Wang et al., Proc.Natl. Acad. Sci. U.S.A. 105:3690-3695 (2008), which is herebyincorporated by reference in its entirety). Although Wang has preparedMan₉ dendrimers which bind 2G12 with K_(D)'s down to 3.1 nM, that levelof binding was achieved only with 9- and 27-mers, whereas 610 nM bindingwas observed with trivalent Man₉ dendrimers. Taken together, these dataindicate that the clustering and/or support of Man₉ by neighboringelements in the glycopeptides obtained in the preceding Examples resultsin better mimicry of the 2G12 epitope than previous Man₉ presentations.These evolved glycoclusters are extremely interesting candidates for invivo immunogenicity studies.

Discussion of Examples 1-4

The preceding Examples demonstrate the in vitro selection of multivalentglycopeptides from diverse libraries (˜10¹³ sequences). It has beenshown that the use of higher temperature in the target binding step ofselection favors glycopeptides with lower multivalency, an effect whichparallels that which was observed in the SELMA selection of glycosylatedDNAs (MacPherson et al., Angew. Chem. Int. Ed. 50:11238-11242 (2011);Temme et al., Chem. Eur. J. 19:17291-17295 (2013); and Temme et al., J.Am. Chem. Soc. 136:1726-1729 (2014), which are hereby incorporated byreference in their entirety). This approach can be used to designmultivalent carbohydrate vaccines targeting additional HIV or cancerepitopes, as well as multivalent carbohydrate ligands for other lectins.The glycopeptides and other conjugates thus obtained will be usefultools in biological studies and for therapeutic applications.

Example 5—Mutational Analysis of 2G12-Binding Clones

One or more point mutations or truncations were introduced into severalof the clones to assess their effects on 2G12 binding. The mutatedsequences, generated according to the procedures described in thepreceding Material & Methods section, are shown in Table 6 below.

TABLE 6 Results of Mutational Analysis SEQ ID K_(D) F_(max) NameSequence^(a) NO: [nM]^(b) [%]^(c) 10F5-C10, 25SXSPHLPVLLSKXVLNDGRRIVQXSSELPXVRRS 52  2.5 nM + 94  0.5 10V1-C7S^(d)XATKTNSKREKTXDNHVTIXRSIPWYTYRWLPN 53  5.6 nM + NA^(d)  0.5 10V1-C7A^(d)XATKTNAKREKTXDNHVTIXRSIPWYTYRWLPN 54  4.4 nM + NA^(d)  0.3 10V1-Δ19XRSIPWYTYRWLPN 55 ~7.5 nM + 20  3.4 10F2-Δ11 XXAALKXQVTDXYALALFHRIL 560.89 nM + 70 0.12 10F3-Δ11 XPNCITQQDVRXTSIPYTYTWP 57   25 nM + 32  4.410F5-Δ11 XVLNDGRRIVQXSCELPXVRRS 58 >200 nM 17 10F12-C2, 21SXSYVTVIPAXNXPEARLGIVSHXPGIRRGKALY 59  186 nM + 90  102 10F3-C15SXDTLHLKQIGGXPNSITQQDVRXTSIPYTYTWP 62 2.91 nM + 63 0.18 ^(a)All peptidesequences used in the 2G12-binding assay were preceded by an N-terminalformyl group and followed by a linker, a His₆-tag and a FLAG-tag(GSGSLGHHHHHHRDYKDDDDK, SEQ ID NO: 1) for purification and radiolabelingpurposes. “X” denotes potential Man₉-glycosylation sites encoded by theAUG codon. ^(b,c)In the assay, the peptides were radiolabeled witheither ³⁵S-cysteine or ³H-histidine as noted above, and incubated withvarious concentrations of 2G12, and 2G12-peptide complexes were isolatedwith magnetic protein G beads. Percentages of the fractions bound werecalculated from radioactivity measured by liquid scintillation countingas described above. K_(D) and F_(max) were calculated by fittingF_(bound) = (F_(max) [2G12])/(K_(D) + [2G12]) to average data points.Errors reported are the standard error of the curve fit. ^(d)10V1-C7sand 10V1-C7A sequences were evaluated by a biolayer interferometry (BLI)assay, rather than the radioactive binding assay, and the material usedwas prepared synthetically rather than by ribosomal translation.

Based on these results, Cys residues can be mutated to Ser or Ala whenthe Cys residue is not involved in disulfide bond formation with asecond Cys residue. Thus, Cys substitution is sequence dependent. Thisis confirmed by 10F12 as compared to 10F5 and 10F3, where Cyssubstitutions were tolerated in the latter but not the former.

Truncation is also feasible where the truncation either does not involveresidues involved in forming the glycosylated epitope recognized by 2G12or residues involved in stabilizing the structure of theglycopolypeptide. This is confirmed by 10F2-A11 and 10V1-A19, whichstill exhibit tight binding to 2G12.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed:
 1. A glycopolypeptide comprising 10 to 80 amino acids,said amino acids including one to ten modified amino acid residueshaving a sidechain comprising a branched oligosaccharide comprising ninemannose moieties, and said amino acids including the amino acid sequenceof XxSIPxYTY (SEQ ID NO: 2) wherein each x at positions 2 and 6 isoptional and can be any natural amino acid, and X at position 1 is oneof said modified amino acid residues, wherein the glycopolypeptide bindsspecifically to carbohydrate-binding monoclonal antibody 2G12 with aK_(d) of less than 100 nM and wherein each modified amino acid residuecomprises a linker molecule between the polypeptide chain and thebranched oligosaccharide, the linker molecule comprising

wherein each of R₁ and R₂ is optionally a direct link or independentlyselected from the group consisting of a linear or branched C₁ to C₁₈hydrocarbon that is saturated or mono- or poly-unsaturated, optionallyinterrupted by one or more non-adjacent —O—, —C(═O)—, or —NR₄—; asubstituted or unsubstituted C₃ to C₁₀ cycloalkanediyl, a substituted orunsubstituted aryl diradical; a substituted or unsubstituted heteroaryldiradical; a monosaccharide diradical; and a disaccharide diradical; R₃is optional and can be —O—, —S—, or —NR₄—; and R₄ is H or a C₁ to C₁₀alkyl.
 2. The glycopolypeptide according to claim 1, wherein theglycopolypeptide comprises from 15 to 70 amino acids.
 3. Theglycopolypeptide according to claim 1, wherein the glycopolypeptidecomprises from 2 to 5 of said modified amino acid residues.
 4. Theglycopolypeptide according to claim 1, wherein the glycopolypeptidebinds specifically to the carbohydrate-binding monoclonal antibody withan affinity that is substantially the same as or lower than the affinityof the carbohydrate-binding monoclonal antibody to its naturallyoccurring binding partner.
 5. The glycopolypeptide according to claim 1,wherein the carbohydrate-binding monoclonal antibody is neutralizingagainst a pathogen.
 6. The glycopolypeptide according to claim 1,wherein said glycopolypeptide comprises one of the following sequences:Sequence SEQ ID NO: XDTLHLKQIGGXPNCITQQDVRXTSIPYTYTWP  3XRSTLNSLEYRXQYATEDPRIRXASIPYTYWWP  5 ATKTNCKREKTXDNHVTIXRSIPWYTYRWLPN  6XATKTNFKREKTXDNHVTIXRSIPWYTYRWLPN  7 XATRTNCKREKTXDNHVTIXRSIPWYTYRWLPN 8 XATKTSCKREKTXDNHVTIXRSIPWYTYRWLPN  9XVLPTIISTNVNPFRXLSIPTYTYLXPITWGEI 10 XTSIPYTYLNRSLWTNYRVNSWSXSKNVNVXPL11 XERPSLXCGLSXLTSGGTQSSVXRSIPFYTYWW 12XATKTNSKREKTXDNHVTIXRSIPWYTYRWLPN 53 XATKTNSKREKTXDNHVTIXRSIPWYTYRWLPN54 XRSIPWYTYRWLPN 55 XDTLHLKQIGGXPNSITQQDVRXTSIPYTYTWP 62

wherein each X is one of said modified amino acid residues to which thebranched oligosaccharide is linked.
 7. A glycopolypeptide comprising 10to 80 amino acids, said amino acids including one to ten modified aminoacid residues having a sidechain comprising a branched oligosaccharidecomprising nine mannose moieties, wherein the glycopeptide bindsspecifically to carbohydrate-binding, neutralizing monoclonal antibody2G12 with a K_(d) of less than 100 nM and wherein the glycopolypeptidecomprises the amino acid sequence: Sequence SEQ ID NO:XLLKXVDQSRLXPVPGIGVTLHXRSIPYSYLPI 4 XHPYNTSRTSAXXAALKXQVTDXYALALFHRIL 13XSPHLPVLLCKXVLNDGRRIVQXSCELPXVRRS 14 XLXFIRIYPTRXQYVYHAPLLTXVRXSPTGPLI15 XCYVTVIPAXNXPEARLGIVCHXPGIRRGKALY 16XSPHLPVLLSKXVLNDGRRIVQXSSELPXVRRS 52 XXAALKXQVTDXYALALFHRIL 56

wherein each X is one of said modified amino residues to which thebranched oligosaccharide is linked.
 8. An immunogenic conjugatecomprising a glycopolypeptide according to claim 1 covalently ornon-covalently bound to an immunogenic carrier molecule.
 9. Theimmunogenic conjugate according to claim 8, wherein the immunogeniccarrier molecule is selected from the group consisting of bovine serumalbumin, chicken egg ovalbumin, keyhole limpet hemocyanin, tetanustoxoid, diphtheria toxoid, thyroglobulin, a pneumococcal capsularpolysaccharide, CRM 197, and a meningococcal outer membrane protein. 10.A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a glycopolypeptide according to claim
 1. 11. Thepharmaceutical composition according to claim 10, wherein theglycopolypeptide is present in the form of an immunogenic conjugatecomprising the glycopolypeptide covalently or non-covalently bound to animmunogenic carrier molecule.
 12. The pharmaceutical compositionaccording to claim 10 further comprising an adjuvant.
 13. Thepharmaceutical composition according to claim 12, wherein the adjuvantcomprises aluminum hydroxide, aluminum phosphate, aluminum potassiumsulfate, beryllium sulfate, silica, kaolin, carbon, water-in-oilemulsions, oil-in-water emulsions, muramyl dipeptide, bacterialendotoxin, lipid, Quil A, non-infective Bordetella pertussis, QS-21,monophosphoryl lipid A, an alpha-galactosylceramide derivative, orPamCys-type lipids.
 14. A glycopolypeptide comprising one or moremodified amino acid residues having a sidechain linked to a branchedoligosaccharide comprising nine mannose moieties, wherein theglycopolypeptide binds specifically to monoclonal antibody 2G12 with aK_(d) of less than 100 nM and wherein said glycopolypeptide comprisesone of the following sequences: Sequence   SEQ ID NO:XDTLHLKQIGGXPNCITQQDVRXTSIPYTYTWP 3 XLLKXVDQSRLXPVPGIGVTLHXRSIPYSYLPI 4XRSTLNSLEYRXQYATEDPRIRXASIPYTYWWP 5 ATKTNCKREKTXDNHVTIXRSIPWYTYRWLPN 6XATKTNFKREKTXDNHVTIXRSIPWYTYRWLPN 7 XATRTNCKREKTXDNHVTIXRSIPWYTYRWLPN 8XATKTSCKREKTXDNHVTIXRSIPWYTYRWLPN 9 XVLPTIISTNVNPFRXLSIPTYTYLXPITWGEI 10XTSIPYTYLNRSLWTNYRVNSWSXSKNVNVXPL 11 XERPSLXCGLSXLTSGGTQSSVXRSIPFYTYWW12 XHPYNTSRTSAXXAALKXQVTDXYALALFHRIL 13XSPHLPVLLCKXVLNDGRRIVQXSCELPXVRRS 14 XLXFIRIYPTRXQYVYHAPLLTXVRXSPTGPLI15 XCYVTVIPAXNXPEARLGIVCHXPGIRRGKALY 16XSPHLPVLLSKXVLNDGRRIVQXSSELPXVRRS 52 XXAALKXQVTDXYALALFHRIL 56XDTLHLKQIGGXPNSITQQDVRXTSIPYTYTWP 62

wherein each X is one of said modified amino acid residues to which thebranched oligosaccharide is linked.
 15. An immunogenic conjugatecomprising a glycopolypeptide according to claim 14 covalently ornon-covalently bound to an immunogenic carrier molecule.
 16. Theimmunogenic conjugate according to claim 15, wherein the immunogeniccarrier molecule is selected from the group consisting of bovine serumalbumin, chicken egg ovalbumin, keyhole limpet hemocyanin, tetanustoxoid, diphtheria toxoid, thyroglobulin, a pneumococcal capsularpolysaccharide, CRM 197, and a meningococcal outer membrane protein. 17.A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a glycopolypeptide according to claim
 7. 18. Thepharmaceutical composition according to claim 17, wherein theglycopolypeptide is present in the form of an immunogenic conjugatecomprising the glycopolypeptide covalently or non-covalently bound to animmunogenic carrier molecule.
 19. The pharmaceutical compositionaccording to claim 17 further comprising an adjuvant.
 20. Thepharmaceutical composition according to claim 19, wherein the adjuvantcomprises aluminum hydroxide, aluminum phosphate, aluminum potassiumsulfate, beryllium sulfate, silica, kaolin, carbon, water-in-oilemulsions, oil-in-water emulsions, muramyl dipeptide, bacterialendotoxin, lipid, Quil A, non-infective Bordetella pertussis, QS-21,monophosphoryl lipid A, an alpha-galactosylceramide derivative, orPamCys-type lipids.